Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element
© Mertz et al; licensee BioMed Central Ltd. 2009
Received: 07 August 2008
Accepted: 03 February 2009
Published: 03 February 2009
Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).
MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.
These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.
Mouse mammary tumor virus (MMTV) is a betaretrovirus that encodes three accessory and regulatory proteins, a superantigen (Sag) [1–3], a dUTPase  and an RNA export protein, Rem . Rem is a 33 kDa protein that is encoded by a doubly spliced mRNA [5, 6]. The N-terminal portion of Rem contains nuclear and nucleolar localization signals as well as an arginine-rich motif similar to the RNA export proteins, Rev, Rex, and Rec, produced by the complex retroviruses, human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type-K (HERV-K), respectively [5, 6]. Our previous data have shown that Rem is larger than other retroviral export proteins due to a unique C-terminus, which negatively regulates Rem-mediated RNA export activity . Negative regulation of MMTV transcription also occurs during viral replication in several cell types [7–10]. MMTV has a complex life cycle that allows transmission through maternal milk to susceptible offspring using dendritic cells as well as B and T cells . Amplification of MMTV in various lymphoid cell types requires virally encoded Sag to effectively transfer virus from lymphocytes to mammary epithelial cells during puberty [12, 13]. Both the sophisticated mode of transmission and production of multiple accessory and regulatory proteins imply that MMTV is a complex retrovirus .
MMTV may interact with human complex retroviruses. Multiple laboratories previously have reported that MMTV sequences are detectable in human breast cancer or lymphomas, but not most normal tissues, using PCR to amplify one or more regions of the viral genome [14–18]. However, not all studies agree [19, 20]. Recent data indicate that MMTV can infect and integrate into chromosomal DNA of cultured human cells [21, 22], suggesting that zoonotic infections can occur. Furthermore, MMTV is highly related to HERV-Ks [also known as human MMTV-like proviruses (HMLs)] . Some intact HERV-K/HML-2 proviruses have been described, consistent with their relatively recent acquisition in the human genome, yet none of these proviruses are known to be infectious [24–26]. A number of HERV-Ks are highly expressed in specific tissues [23, 27]. In addition, a recent report indicates that antibodies to HERV-K/HML-2 are detectable in the plasma of breast cancer and lymphoma patients, and these titers dropped when the cancers were treated. HERV-K reverse transcriptase activity, viral RNA, processed viral proteins, and virus-like particles also could be detected in patient plasma . Together, these experiments suggest that sporadic MMTV infections of human cells may result in interactions with HERV-Ks or the generation of recombinant infectious viruses.
Prior experiments indicate that HIV Rev and HTLV Rex can activate expression from reporter plasmids containing the HERV-K Rec-responsive element (RcRE) . Because of sequence and organizational similarities between MMTV and HERV-K and the potential for MMTV infection of human cells, we have tested for interactions between heterologous retroviral export proteins and the Rem-responsive element (RmRE) using our previously described reporter vector, pHMRluc . Surprisingly, Rev and Rex, but not Rec, could activate MMTV-based reporter gene expression in human T cells and was dependent on the presence of the RmRE. Cell fractionation experiments followed by RNA quantitation suggested that each of the regulatory proteins, including Rem, did not affect RNA export or stability using an MMTV-based reporter vector. Rem activity was undetectable using heterologous response elements. These results suggest that retroviral export elements recognize similar features of RNA structure and support the idea that MMTV is a complex murine retrovirus that may interact with other retroviruses in human cells.
Additional experiments then were performed in Jurkat cells using the MMTV-based reporter vector (pHMRluc) and co-transfected expression vectors for GFP-tagged Rem, Rev, or Rex. RNAs from cytoplasmic and nuclear fractions were treated with DNase I and subjected to semi-quantitative RT-PCRs using primers specific for the Renilla luciferase gene or gapdh  (Figure 7C). PCRs without added reverse transcriptase showed that DNA contamination was absent (data not shown). Cytoplasmic RNA levels then were quantitated using ImageJ software after normalization for gapdh expression. Unexpectedly, these experiments indicated that Rem, Rev, or Rex had little effect on the levels of RNA in the nucleus or cytoplasm in Jurkat cells, suggesting that these proteins do not affect intron-containing transcript stability or export (Figure 7D).
Our previous experiments have shown that MMTV Rem functions in nuclear export of unspliced viral RNA in rodent cells . In this manuscript, we have shown that Rem functions in human cell lines. Our results also indicate that Rev and Rex can increase reporter gene expression by interaction with the MMTV RmRE in human Jurkat T cells (Figures 2 and 4). Rex also could function on the RmRE in 293T HEK cells. Prior data indicate that some retroviral export proteins function on heterologous retroviral RNAs. For example, Rex can bind and function on both the RRE and the RxRE [43, 44]. However, the interaction is not reciprocal since Rev cannot act on the RxRE . In this respect, the RmRE is quite permissive since it is required for enhancement of luciferase activity by Rem, Rev and Rex in human T cells. No effect of Rem was observed on the HIV and HTLV response elements (Figure 8). Surprisingly, the Rec export protein from the human retrovirus most closely related to MMTV, HERV-K/HML, had no effect on expression from the MMTV RmRE (Figure 6). Further, no effect of Rem was observed on the RcRE, although both HIV Rev and HTLV Rex have been reported to increase expression from the HERV-K response element . However, polymorphisms have been observed in different HERV-K proviruses , and it is possible that other RcRE variants might function with MMTV Rem or that other Rec variants may be functional on the RmRE. Given that the regulatory proteins require formation of specific secondary structures rather than a simple primary sequence [29, 45–48], Rec also may need secondary or tertiary structures not found in the RmRE.
The effect of Rev and Rex on the MMTV RmRE appears to be specific in human cells by several criteria. First, increases in reporter gene activity that were dependent on the RmRE were only observed in human cells with Rex or Rev. Although different results have been reported [36, 49], Rev appears to function in both mouse and human cell lines using vectors with a design similar to that of pHMRluc, but based on the 3' end of the HIV genome . Rex also has been reported to function in both human and mouse cells , although Rev and Rex have primarily been tested in fibroblasts [36, 50], which are not natural target cells for HIV, HTLV or MMTV. Second, a Rev mutant defective in the nuclear export sequence gave no specific effect in the pHMRluc assay, similar to the effect observed with RRE-containing vectors . Third, a dominant-negative mutant nucleoporin in the Crm1 pathway inhibited Rem, Rev, and Rex activation of reporter expression through the RmRE in human cells. Rem previously has been shown to require Crm1 in rodent cells , whereas Rev and Rex use Crm1 in human cells [33, 41]. Fourth, no effect of Rec was observed on the RmRE in either mouse or human cells. Fifth, insertion of different response elements in the pHMRluc vector yielded the expected increases in luciferase activity after expression of the homologous export protein. These results indicate that the MMTV-based vector allows the activity of other response elements and that each of the GFP-fusion proteins is functional (Figure 8). Although we may have lowered the sensitivity for detection of regulatory protein function in mouse cells by testing fusion proteins, Western blotting using an antibody that recognizes all of the fusion proteins allowed us to verify that similar amounts of each protein were expressed in transfection assays. Prior experiments by Dangerfield et al. suggest that Rev can bind to the MMTV LTR and stimulate luciferase expression from constructs containing the MMTV LTR in monkey cells . Our studies differ significantly since their data were obtained by insertion of MMTV sequences into an HIV-based vector, and the ability of Rem to function on heterologous response elements was not determined. Furthermore, only the MMTV LTR, which lacks a portion of the RmRE  (our unpublished data), was present in the HIV vector . Thus, our data argue for a specific effect of HIV and HTLV regulatory proteins on the MMTV RmRE in human cells.
Previous experiments from our laboratory have shown that human Jurkat T cells can produce mature MMTV particles after transfection of a cloned provirus, and these particles are infectious for mice [52, 53]. Consistent with this observation, our current data indicate that Rem can function in human cells. The reports of MMTV infection of human cells and detection of MMTV sequences in breast cancers and lymphomas [14–18] appear to be feasible since most steps of viral replication occur in human cells. Cell entry would provide the primary barrier to infection . Although human cell infections appear to be inefficient and infrequent, certain cell types may have an additional entry receptor, which is dependent on cellular activation or differentiation state. The ability of Rev and Rex to function on the MMTV RmRE in human T cells suggests that rare interactions of these viruses could occur.
Rev is known to have multiple functions, including enhancement of RNA encapsidation of HIV and SIV-based vectors . Our previous results indicated that export of unspliced MMTV RNA and Gag expression from a transfected MMTV provirus requires Rem in rat fibroblast cells ; encapsidation was not measured. The reporter vector pHMRluc is based on the 3' end of the MMTV provirus and has been shown to be responsive to Rem only in the presence of the RmRE in rat, mouse, and human cells  (this study). Further, the use of the Renilla luciferase gene in the vector provides both a sensitive and highly quantitative assay, which is difficult to achieve using RNA fractionation experiments and Northern blotting. Rev/RRE interactions also have been shown to affect Gag trafficking and HIV assembly, and it has been suggested that export elements facilitate "marking" of RNAs in the nucleus for particular events in the cytosol . Our experiments show that Rev and Rex function through Crm1 on pHMRluc (Figure 9). Nevertheless, cell fractionation experiments with the pHMRluc vector indicate that the regulatory proteins primarily lacked effects on nuclear RNA export and RNA stability (Figure 7). Since effects on cytosolic RNA levels and Gag production were clearly demonstrable using an MMTV proviral clone with a transposon insertion into the rem coding sequence , our results with the pHMRluc vector suggest that different sequence elements at the 5' end of the full-length MMTV RNA allow additional effects of Rem on RNA stability and/or export.
Published experiments indicate a wide variability (0 to 10-fold) in Rev function on RNA export [55, 57–60]. Suboptimal splicing appears necessary to allow the accumulation of genomic HIV RNA and the export effects of Rev . Efficiency of splicing of full-length MMTV RNA versus pHMRluc vector RNA appears to be an unlikely explanation for differences in observed nuclear export. The splice donor and acceptor sites found in pHMRluc are those normally used to generate either the rem or sag fully spliced mRNAs, and the low abundance of these RNAs in MMTV-infected cells [6, 62] suggests that splicing at these sites is suboptimal compared to those used to produce MMTV env RNAs from genomic RNAs. Rev also appears to overcome effects of several cis-acting repressive sequences, including sequences within gag-pol [63, 64] as well as env sequences that overlap with the RRE [65, 66]. The repressive sequences in HIV gag-pol appear to be AU-rich, and mutation led to increased steady state RNA levels . The pHMRluc vector lacks gag-pol sequences (Figure 1), but our previous work with Rem-deficient MMTV genomic clones was consistent with defective RNA export, rather than a stabilization effect.
The cell fractionation data with pHMRluc (Figure 7) and MMTV genomic length RNA  argue that Rem has multiple functions, including both export and post-export activities. Rev and Rex have been reported to function at the level of translation [59, 67, 68]. Specific cis-acting elements found in the RU5 and gag regions of several retroviruses appear to affect translation [69–73], but such sequences are absent in the pHMRluc vector. Since the post-export function of Rem with pHMRluc is sensitive to competition with a Crm1-binding site on Nup214 (ΔCAN) (Figure 9), it is possible Crm1 dictates Rem protein export independent of the vector RNA. Nevertheless, Rem binding to the 3' RmRE, perhaps in the cytoplasm or after binding of a cellular protein in the cytoplasm, may promote a post-export step, such as translation. Rem binding through sequence elements at the 5' end of the MMTV RNA may increase Crm1-dependent export, but such 5' elements may not be necessary for detection of the post-export activity of the pHMRluc vector. Our current data indicate that the RmRE maps to the junction of the envelope gene and the 3' LTR using deletion analysis with the pHMRluc vector and co-transfection of a Rem expression vector (see below). Interestingly, these results suggest that all MMTV mRNAs contain the 3' RmRE, unlike the RRE, which would be removed from completely spliced HIV mRNAs, such as those encoding Tat, Nef, and Rev . Previously published data indicate that export of unspliced genomic MMTV RNA, but not partially spliced envelope RNA, is leptomycin B and, by implication, Crm1-dependent . Such experiments suggest that only unspliced MMTV RNA is selectively exported. Therefore, it is possible that the MMTV genome contains two RmREs, one at the 5' end of viral RNA present only in unspliced RNA and a second element at the 3' end present in all MMTV RNAs. The 3' element may facilitate translation of all mRNAs, whereas the 5' element would specifically facilitate nuclear export of genomic RNA. Cell-type specific effects also may occur. Characterization of the molecular mechanisms of Rem function will require further investigation.
Both the pHMRluc vector and MMTV genomic RNAs contain a RmRE that spans the envelope-LTR junction  (Mertz et al., in preparation). Published data indicate that retroviral export/regulatory proteins bind to complex RNA structures that have multiple stems and loops [29, 48, 75, 76]. Rev and Rec appear to bind to RNA stems with a bulge, and recognition of heterologous elements may not occur through the same primary sequence as the homologous protein . Our current data using RmRE susceptibility to several RNases is consistent with a complex structure containing multiple stems and bulges, which encompasses a region of ca. 500 bases (Mertz et al., in preparation) rather than the single stem with multiple bulges previously proposed . The export of unspliced retroviral RNA is known to require specific cellular proteins, such as hnRNPs and Sam68 [77, 78], and binding of these cellular proteins may determine the cell-type specificity observed in our experiments. Since retroviral export/regulatory proteins recognize certain RNA secondary structures [48, 79], one or more of these proteins may bind to and function on specific cellular RNAs as reported for Rex .
RNA-binding proteins appear to regulate several steps following transcription, leading to coordinated regulation of cellular RNAs with related functions called RNA regulons . MMTV replication in the mouse requires several different cell types, including lymphocytes and mammary epithelial cells . We previously have shown that MMTV replication is controlled at the transcriptional level during mammary gland development coordinately with several milk-specific genes [82, 83]. Therefore, post-transcriptional control of MMTV expression also may be modulated by Rem depending on the cell type and state of differentiation. Our results provide additional evidence that MMTV is a murine complex retrovirus with the potential to interact with human retroviruses .
Cell lines and transfections
Jurkat human T lymphoma cells were maintained in RPMI media supplemented with 5% fetal calf serum (FCS), gentamicin sulfate (50 μg/ml), penicillin (100 U/ml) and streptomycin (50 μg/ml). HC11 normal murine mammary epithelial cells were maintained in RPMI supplemented with 10% FCS, gentamicin sulfate (50 μg/ml), penicillin (100 U/ml), streptomycin (50 μg/ml), insulin (0.5 μg/ml) and epidermal growth factor (0.5 μg/ml). The 293T human embryonic kidney cells were grown as previously described  in Dulbecco's modified Eagle's medium containing 7.5% fetal bovine serum and antibiotics.
Jurkat cells were transfected by electroporation using a BTX ECM600 instrument. Cells (1 × 107) were mixed with the appropriate plasmid DNA in a volume of 400 μl RPMI medium prior to electroporation in 4 mm gap cuvettes (260 V, 1050 μF and 720 ohms). Transfected cells then were incubated at 37°C in complete medium and harvested two days after transfection for Western blotting and reporter assays. HC11 cells also were transfected by electroporation using a BTX electroporator. Cells (1 × 107) were mixed with the appropriate plasmid DNA in a volume of 200 μl of RPMI prior to electroporation in 2 mm gap cuvettes at 140 V, 1750 μF, and 72 ohms. The 293T cells were transfected essentially as described  by the calcium phosphate method. On the day before transfection, 5 × 105 cells were added to each well of a 6-well plate, and DNA (total of 6 μg) in 0.25 M CaCl2 (100 μl) was added dropwise to 100 μl of 2× HBS (280 mM NaCl, 10 mM KCl, 1.5 mM disodium phosphate, 12 mM dextrose and 50 mM HEPES, pH 7.05) with vortexing. The precipitate was allowed to form at room temperature for 10 to 15 minutes, and the solution was added dropwise to the cells in growth medium. Cells then were incubated at 37°C from 4 to 8 hours, the medium was removed, and cells were washed in phosphate-buffered saline prior to replacement with fresh growth medium. Transfected cells were harvested after two days and assayed for reporter gene levels and protein expression. All transfections were performed in triplicate and contained the same total amounts of plasmid DNA. A constant amount of pGL3 control containing the firefly luciferase gene was included in each transfection to normalize for any differences in DNA uptake. Some experiments also tested for DNA uptake after determination of the percentage of cells expressing a GFP control vector using FACS analysis. No significant differences were observed using either of the two methods. All reported experiments were repeated at least twice with similar results.
The RemGFP, HMRluc and HMΔeLTRluc plasmids have been described . The plasmid EGFPN3 was obtained from Clontech, and pGL3-Control plasmid was obtained from Promega. The expression plasmid for the Δ3 mutation in the Rev nuclear export sequence was received from Dr. Tom Hope. The pcΔCAN (dominant-negative Nup214) and pcTapA17 (dominant-negative Tap/NXF1) expression plasmids were kindly provided by Dr. Bryan Cullen (Duke University). The empty vector pBC12/CMV was obtained by excision of the ΔCAN cDNA from pcΔCAN. The pRRERluc plasmid was constructed by insertion of the HIV-1 RRE, amplified from the pDM128 vector (provided by Dr. Tom Hope), into an engineered ScaI site downstream of the splice acceptor site and upstream of the SV40 poly(A) signal in HMΔeLTRluc. The plasmids RxRE1Rluc and RxRE2Rluc were generated by amplification of RxRE1 and RxRE2 from pcgagRxREI and pcgagRxRE2, respectively (provided by Dr. Pat Green) and insertion into an engineered ScaI site downstream of the splice acceptor site and upstream of the SV40 poly(A) signal in pHMΔeLTRluc. The RcRERluc plasmid was made by amplification of the RcRE from pJY76 (provided by Dr. Bryan Cullen) and insertion into an engineered ScaI site downstream of the splice acceptor site and upstream of the SV40 poly(A) signal in pHMΔeLTRluc. RevGFP, Rex1GFP, Rex2GFP and RecGFP were generated by cloning of the individual cDNAs in-frame with a C-terminal GFP tag in the vector EGFPN3.
Luciferase assays were performed using the dual-luciferase reporter assay system (Promega) to quantitate both Renilla and firefly luciferase activities .
Northern blotting and RT-PCRs
RNA was extracted from transfected Jurkat cells as described previously , except that the lysis buffer (10 mM Tris-HCl, pH 8.0, 140 mM NaCl, 1.5 mM MgCl2, 20% glycerol) contained 0.1% NP-40 rather than 0.5% NP-40. Lysis buffer was supplemented with 10 mM vanadyl ribonucleoside complexes (New England Biolabs) to inhibit ribonucleases prior to use. Cells were mixed using a vortex mixer, examined for lysis by microscopy, and nuclei were pelleted by centrifugation (300 × g for 5 minutes at 4°C). The supernatant (cytoplasmic fraction) was removed and again subjected to centrifugation (1,200 × g for 5 minutes). The cytoplasmic fraction was then subjected to centrifugation at 8,000 × g for 5 minutes at 4°C. The nuclear pellet was washed once with lysis buffer, and the supernatant containing residual cytoplasm discarded. Nuclear samples were processed in Tri-Reagent (2 M guanidine isothiocyanate, 12.5 mM sodium citrate, pH 7.0, 0.25% Sarkosyl, 0.05 M 2-mercaptoethanol, 0.2 M sodium acetate, pH 5.2, and 50% water-saturated phenol, pH 7.5), whereas Tri-Reagent LS (Molecular Research Center, Inc.) was used for cytoplasmic fractions. Samples then were processed as described by the manufacturer. RNAs were precipitated using ethanol, washed in 70% ethanol, and precipitates were collected by centrifugation at 10,000 × g for 30 minutes at 4°C. DNA was removed after precipitation of high-molecular-weight RNA in 3 M sodium acetate , pellets were washed in 70% ethanol, and the quantity of the RNA was determined by absorbance readings at 260 nm. Procedures for Northern blotting using formaldehyde-containing agarose gels have been described . To test for the integrity of the cellular fractionation, each lane of the gel contained 10 μg of fractionated RNA prior to electrophoresis and transfer to Hybond N+ nylon membranes in 0.15 M sodium citrate and 1.5 M NaCl. RNA samples then were cross-linked to the membrane using UV light and stained with methylene blue prior to photography.
Fractionated RNAs from transfected cells also were used for RT-PCRs. Each RNA sample (1 μg) was digested with 1 U of DNase I (amplification grade, Invitrogen) in the presence of 0.5 μl of RNaseOUT (Invitrogen) ribonuclease inhibitor for 15 minutes at room temperature. The reaction was terminated by the addition of EDTA to 2.5 mM and incubation for 10 minutes at 65°C. The treated RNAs then were incubated with 50 pmol oligo(dT)17 primer and 1 mM deoxyribonucleoside triphosphates for 5 minutes at 65°C and then quickly cooled on ice for 5 minutes. Subsequently, first-strand buffer (Invitrogen) was added in the presence of 10 mM DTT, 20 U RNaseOUT, and 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen) in a 20 μl reaction. Samples were incubated for 50 minutes at 37°C and then terminated by heating at 70°C for 15 minutes. PCRs were performed using 1 μl of the cDNA reaction, 25 pmol of each primer, 0.2 mM deoxyribonucleoside triphosphates, 20 mM Tris-HCl pH 8.55, 2.5 mM MgCl2, 16 mM ammonium sulfate, 100 ug/ml BSA, and 2.5 U of KlenTaq (Sigma Aldrich) in a reaction volume of 50 μl. Samples were subjected to 3 minutes at 94°C for 3 minutes followed by 35 cycles consisting of incubations at 94°C for 1 minute, 50°C for 45 seconds, and 72°C for 45 seconds. The primers used to detect unspliced RNAs containing the Renilla luciferase gene were Rluc1409(+) (5' GAT TGG GGT GCT TGT TTG G 3') and Rluc1904(-) (5' TTC CCA TTT CAT CAG GTG C 3'). Primers for gapdh have been described . Similar reactions for cat-specific transcripts contained 5 μg RNA and 1.5 U DNase I, and 3.5 μg of the treated RNA was used to make cDNA in 20 μl and then diluted two-fold. Either 2 μl or 4 μl of the diluted cDNA was used in 50 μl PCRs containing cat primers [186(+) (5' TCT TGC CCG CCT GAT GAA TGC 3') and 653(-) (5' CCG CCC TGC CAC TCA TCG CAG 3')] and REDTaq mix (Sigma-Aldrich). PCR samples were analyzed on 1.5 or 2% agarose gels and stained with ethidium bromide prior to photography.
Antibodies and Western blotting
Western blot assays were performed essentially as described previously . Transfected cells were harvested to obtain whole-cell extracts by addition of one volume of 250 mM Tris-HCl, pH 6.8, 20% glycerol, 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, and 0.2% bromophenol blue to cells in one volume of phosphate-buffered saline (PBS) followed by boiling for 5 minutes. Proteins were resolved on 10 or 12% polyacrylamide gels containing 1% SDS and transferred to a nitrocellulose membrane. Membranes were blocked with 5% milk in Tris-buffered saline Tween 20 (TBST; 20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20) for 1 hour. The primary antibody was diluted in TBST containing 5% milk and incubated with the membrane for 1 hour followed by three washes in TBST for 5 to 10 minutes each. The horseradish peroxidase-conjugated secondary antibody was diluted in TBST containing 1% milk and incubated with the membrane for 45 minutes prior to three additional 5 to 10 minute washes. All steps were performed at 25°C with shaking. Western Lightning enhanced chemiluminescent reagent (Perkin-Elmer) was used to detect antibody binding. Monoclonal antibodies specific for actin (Calbiochem) or GFP (Becton Dickinson) were used at a dilution of 1:10,000 or 1:8000, respectively.
This work was supported by NIH grant R01 CA116813. We thank Tom Hope, Bryan Cullen, and Patrick Green for reagents as well as Jon Huibregtse, Rick Russell, and members of the Dudley lab for helpful comments.
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