Identification of novel CDK9 and Cyclin T1-associated protein complexes (CCAPs) whose siRNA depletion enhances HIV-1 Tat function
© Ramakrishnan et al.; licensee BioMed Central Ltd. 2012
Received: 19 March 2012
Accepted: 5 October 2012
Published: 30 October 2012
HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5′ end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described – the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression.
We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies.
Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.
A critical step in the HIV-1 replication cycle is transcription of the integrated provirus by RNA polymerase II (RNAP II). Although RNAP II initiates transcription from the HIV-1 LTR at a relatively high basal rate, the polymerase stalls after synthesis of a short transcript due to the action of two negative elongation factors, NELF and DSIF, which associate with the RNAP II complex and inhibit elongation [reviewed in [1–4]]. Pausing of RNAP II elongation is also seen in cellular genes and serves as a quality control mechanism to enable efficient capping of the 5′ end of the mRNA [5, 6]. In the case of HIV-1, the viral Tat protein recruits a cellular protein kinase complex termed P-TEFb to the TAR RNA element that forms at the 5′ ends of the nascent viral transcript. The catalytic component of P-TEFb is composed of CDK9 as the catalytic subunit and a Cyclin regulatory subunit, either Cyclin T1 (CCNT1) or Cyclin T2 (CCNT2). P-TEFb activates transcriptional elongation by phosphorylating the carboxyl terminal domain (CTD) of the large subunit of RNAP II; P-TEFb also phosphorylates specific protein subunits of NELF and DSIF, and this negates their inhibition of elongation. For cellular genes, P-TEFb can be recruited to the RNAP II complex by different mechanisms to stimulate elongation. The bromodomain protein BRD4 is bound to a portion of P-TEFb and directs P-TEFb to actively transcribed genes that are marked by acetylated histones [7–9]. Transcription factors that bind directly to DNA elements in cellular promoters, such as NF-κB, cMyc, and MEF2, can also recruit P-TEFb to active genes [10–12].
P-TEFb is known to exist in three distinct complexes: 1) the “Core” complex composed of CDK9/CCNT1 or CCNT2/BRD4; 2) the “7SK snRNP” complex composed of CDK9/CCNT1 or CCNT2/7SK snRNA/ HEXIM1 or HEXIM2/LARP7/ MEPCE; 3) “Super Elongation Complex” (SEC) composed of CDK9/CCNT1 or CCNT2/ELL2/AFF4/ENL/AF9 . Gel filtration analyses of purified Flag-tagged CDK9 have confirmed that these three complexes exist as distinct biochemical entities . CDK9 kinase activity is repressed in the 7SK snRNP, and this complex is not thought to directly activate transcriptional elongation. Rather, the 7SK snRNP may be a catalytically inactive pool of P-TEFb from which active P-TEFb can be recruited to function in either the Core or SEC P-TEFb complex to activate elongation . Consistent with this idea, disruption of the 7SK snRNP by siRNA depletions of HEXIM1 results in increased levels of Tat activation of the viral LTR .
The HIV Tat protein binds directly to CCNT1 and thereby targets only CCNT1-containing P-TEFb complexes . Tat is capable of utilizing the Core P-TEFb complex by displacing Brd4 from CDK9/CCNT1 . Tat can trigger the release of CDK9/CCNT1 from the 7SK snRNP and utilize this P-TEFb complex [18–20]. Tat also associates with the SEC and stabilizes this P-TEFb complex to activate viral gene expression [21, 22].
Nearly all cellular processes are carried out by multi-protein complexes [23, 24]. A recent study using a high-throughput integrative mass spectrometry-based analysis described the human endogenous complexome – the set of multi-protein complexes in HeLa cells . In this landmark study, 1,796 primary antibodies were used in 3,390 immunoprecipitations of HeLa cell nuclear extracts. Conditions of immunoprecipitations were developed to preserve weak protein interactions and high levels of reciprocity in affinity purifications were used to identify distinct multi-protein complexes. We have mined this data set to identify multi-protein complexes that contain CDK9 and CCNT1. The three known P-TEFb complexes -- Core, 7SK snRNP and SEC -- as well as nine additional CDK9/CCNT1 complexes were identified in this analysis. In this study, we focused on five of these novel complexes and found that siRNA depletion of protein subunits of these novel complexes enhanced HIV-1 Tat function and viral gene expression. Although the mechanisms whereby siRNA disruption of these novel CDK9/CCNT1 complexes stimulate Tat function remain to be elucidated, our data suggest that their disruption may increase the level of P-TEFb available for Tat to utilize, similar to disruption of the 7SK snRNP. Our study indicates that the complexity of P-TEFb is considerably greater than previously appreciated.
Identification of novel CDK9/CCNT1-associated proteins (CCAPs)
A prominent CCAP not shown in Figure 1 due to size constraints contains CDK9/CCNT1 in a very large multiprotein complex that regulates transcription initiation and chromatin remodeling [CBP/p300; the 15 subunit INO80 complex; 20 subunit SWI/SNF complex; 30 subunit Mediator (Mediator has been implicated in HIV-1 replication in siRNA screens ). Two CCAPs involved in transcription elongation are prominent in Figure 1: AFF4/AFF1/MLLT1/ELL2/MLLT3 (Super-Elongation complex D) and WDR82/PPP1R10/TOX3 (complex E) that interacts with the LEO1/PAF complex (histone methyltransferase activity) and is implicated in transcriptional elongation . TTF2 (complex G) is a CCAP involved in RNA Polymerase II termination. Two CCAPs associate with nuclear pores, RANGAP1 (complex H) and TPR (complex I). Other CCAPs included the checkpoint module ATR/ATRIP (complex J), ubiquitin binding protein WRNIP1 (complex K), and SCFFBXO11 ubiquitin E3 ligase (complex L). The proteins PRPF8 (complex F) and RANBP2 (complex H) have been implicated in HIV-1 replication in siRNA screens . The identification of novel CCAPs involved in RNA elongation, RNA splicing, transcription termination, and nuclear pores suggest that the role of CDK9/CCNT1 in cellular and HIV-1 gene expression is considerably more intricate than previously appreciated.
SiRNA depletion of CCAPs
Protein subunits of CCAPs depleted in this study
Function of depleted protein
F-box protein 11
UBR6;VIT1; FBX11; PRMT9; UG063H01
Phosphorylation-dependent ubiquitination, regulates p53 and BCL6
S-phase kinase-associated protein 1
OCP2; p19A; EMC19; SKP1A; OCP-II; TCEB1L
protein phosphatase 1, regulatory subunit 10
FB19; CAT53; PNUTS; PP1R10
Targets protein phosphatase-1 (PP1) to the nucleus, in DNA damage response
WD repeat domain 82
SWD2; MST107; WDR82A; MSTP107; PRO2730; TMEM113; PRO34047
Component of the mammalian SET1A /SET1B histone H3-Lys4-methyltransferase complexes
TOX high mobility group box family member 3
Unwinding of DNA, chromatin structure alteration and neuronal transcription
Translocated promoter region (to activated MET oncogene)
Directly interacts with several components of nuclear pore complexes (NPCs). Nuclear export of mRNAs and some proteins.
Transcription termination factor, RNA polymerase II
Critical role in altering protein-DNA interactions. Has dsDNA-dependent ATPase and RNAPII termination activity. Plays a role in pre-mRNA splicing.
Werner helicase interacting protein 1
WHIP; bA420G6.2; RP11-420G6.2
Interacts with the N-terminal portion of Werner protein containing the exonuclease domain and implicated in Werner’s syndrome. It may influence aging process.
We used the TZM-bl cell line for our depletion experiments; this cell line is a HeLa cell derivative that expresses CD4, CXCR4, and CCR5 on its cell surface and therefore can be infected by HIV-1 . TZM-bl cells also contain an integrated copy of the HIV-1 LTR with a Luciferase reporter protein. Tat transactivation assays can be readily performed in TZM-bl cells by transfection of a Tat expression plasmid and measurement of Luciferase expression from the integrated provirus. As the human complexome data set was generated in HeLa cells , the CCAPs shown in Figure 1 are likely similar if not identical between TZM-bl and HeLa cells.
Because PPR1R10 is associated with Protein Phosphatase 1 and regulates the activity of a cellular enzyme that regulates many cellular processes, we monitored the effect of siRNA depletion of PPP1R10 on cell viability using a Trypan Blue exclusion assay. TZM-bl cells were transfected with siRNAs against PPP1R10 or control siRNAs. Cells were collected at 72 hours post-transfection and divided into two groups -- one group was lysed, and depletion of PPP1R10 was verified by immunoblot (data not shown); and the other group was assayed for cell viability. As shown in Figure 2E, there was only a small reduction in the viability of the cells depleted of PPP1R10 compared to control siRNA-treated cells. This small reduction in cell viability by siRNA depletion of PP1R10 is unlikely to explain the enhancement of Tat function by the depletion (see below).
SiRNA depletion of CCAPs enhances Tat activation of HIV-1 LTR
Based on the results shown in Figure 3, it is conceivable that the five CCAPs examined could function additively to regulate Tat function. Therefore, we evaluated the effects of depletions of combinations of CCAPs on Tat transactivation in TZM-bl cells (Additional file 1: Figure S1). This analysis demonstrated that some combinations showed a strong additive effect on increasing Tat transactivation, especially the combination of TTF2 and FBXO11.
Activation of NF-κB is not responsible for the increase of HIV-1 gene expression upon depletion of CCAPs
Enhancement of Tat activation by CCAP depletions is not the result of increased Tat protein expression or increased release of CDK9/CCNT1 from 7SK snRNP
We also investigated whether depletion of CCAPS might affect the amount of CDK9/CCNT1 in the 7SK snRNP. Depletion of TTF2 and FBXO11 was used for this control experiment because this combination of CCAP depletions showed the greatest level of Tat enhancement observed in this study (see Additional file 1: Figure S1). HeLa cells were first transfected with a combination of TTF2 and FBXO11 siRNAs or control siRNAs, and 48 hours later cells were transfected with expression plasmids for Flag-tagged wild type Tat or mutant Tat and HA-CCNT1; 48 hours later cell extracts were prepared for immunoprecipitations with an antiserum against CDK9. As shown in Figure 5B, no difference in the association of CDK9 with CCNT1 or HEXIM1 was observed in cells transfected with Flag-tagged wild type and mutant Tat and treated with either control or TTF2+ FBXO11 siRNAs. This result suggests that depletion of CCAPs does not affect the level of CDK9/CCNT1 in the 7SK snRNP.
Combination of TTF2 and FBXO11 depletion sensitizes Jurkat CD4+T cells to reactivation of latent HIV-1
The production of cellular mRNA and HIV-1 RNA from the integrated provirus is a highly regulated process in which the individual steps are linked. RNA splicing is coupled to transcriptional elongation , and nuclear export of processed RNA can be affected by splicing . Most, if not all proteins responsible for production of RNA exist in multi-protein complexes, and the understanding of mechanisms involved in HIV-1 RNA production will require the identification of protein complexes involved in the individual steps of viral RNA production. In this study, we mined the recently described HeLa cell nuclear complexome  to investigate multi-protein complexes involved in HIV-1 Tat function and viral gene expression. Our analysis of the complexome data set identified 12 multi-protein complexes which contain CDK9/CCNT1, the enzymatic core of the general RNAP II elongation factor known as P-TEFb that mediates Tat transactivation. Our analysis identified the three previously known CDK9/CCNT1 complexes –Core, 7SK snRNP, and SEC P-TEFb complexes , thereby validating our analytical approach. We identified nine additional multi-protein complexes that contain CDK9/CCNT1; eight of these novel CDK9/CCNT1 complexes are shown in Figure 1, while the largest complex identified (not shown in Figure 1) contains CBP/p300, the 15 subunit INO80 complex, 20 subunit SWI/SNF complex, and 30 subunit Mediator complex. We have termed these multi-protein complexes CCAPs for CDK9/CCNT1-associated protein complexes.
To begin to investigate the role of novel CCAPs in Tat function, we carried out siRNA depletions of protein subunits of five CCAPs. We chose to focus on these five CCAPs in the present study as our initial results indicated that depletions of their protein subunits enhanced Tat function. We found that disruption of these CCAPs by siRNA depletions resulted in enhanced Tat function in TZM-bl cells. Because previous work has shown that depletion of HEXIM1 in the 7SK snRNP enhances Tat function by increasing the available pool of CDK9/CCNT1, we favour the hypothesis that depletion of these five CCAPs enhances Tat function by also increasing the available pool of CDK9/CCNT1. However, future studies will be required to determine the molecular mechanisms whereby disruption of these five CCAPs enhances Tat function. Our initial results with a CCAP containing ATR/ATRIP (Complex J, Figure 1) indicated that depletion of these proteins inhibits Tat function, consistent with a previous study . Additionally, it is likely that depletion of subunits of the largest CCAP (CBP/p300, the 15 subunit INO80 complex, 20 subunit SWI/SNF complex, and 30 subunit Mediator complex) will inhibit Tat, as some of these protein subunits have been shown to play a positive role in Tat function and HIV-1 replication [28, 46, 47].
CCAPs examined in this study
The protein subunits of the CCAPs examined here have been previously shown to be involved in a number of cellular processes of interest to cellular and HIV-1 RNA metabolism. The finding that these proteins associate with CDK9/CCNT1 has a number of intriguing functional and mechanistic implications. Short descriptions of the known functions of the protein subunits of these CCAPs are presented below.
Complex E (Figure 1) recruits the phosphatase PP1 into the nucleus and is involved in the DNA damage response [31, 48]. Additionally, WDR82 is found in the SET1A histone H3-Lys4-methyltransferase complex  and this chromatin modification in associated with actively expressed genes.
This CCAP subunit is involved in the termination of RNAP II transcription . TTF2 has also been implication in mitotic repression of transcriptional elongation . A recent publication reported that TTF2 is associated with decapping factors and the Xrn2 exonuclease at transcriptional start sites and is involved in premature termination of RNAP II transcription .
This CCAP subunit is a large coiled-coiled protein and is localized within the nuclear basket of the nuclear pore complex; TPR plays a role in nuclear export of mRNA [34, 50]. Fusion of the 5′ end of the TPR gene with several different kinase genes is associated with cancer.
This CCAP subunit interacts with the Werner protein and is implicated in Werner’s syndrome . The Werner protein is involved in RNAP II transcription and its dysfunction in Werner’s syndrome results in an accelerated aging phenotype .
Complexity of P-TEFb and CCAPs
The identification of 12 CCAPs in this study and the heterogeneity in the catalytic core of P-TEFb reveal an enormous potential complexity in the biological functions of CDK9 and CCNT1. The catalytic core is composed of a heterodimer of CDK9 and a Cyclin subunit, either CCNT1 or CCNT2 . Additionally, two spliced variants of CCNT2 are expressed, CCNT2a and CCNT2b , as are two isoforms of CDK9, a major 42 kDa isoform and a minor 55 kDa isoform that arises from an upstream transcriptional start site . It is possible that these two CDK9 isoforms may differentially associate with the CCAPs shown in Figure 1, and it is possible that one or both of the CCNT2 isoforms may replace CCNT1 in these multi-protein complexes. Complexes that differ by their CDK9 or Cyclin subunit may have distinct functional properties, as well as distinct expression patterns in different tissues. Transcriptional profiling of cells depleted for either CCNT1 or CCNT2 has identified mRNAs whose expression patterns appear dependent on either CCNT1 or CCNT2 [53, 54]. The expression pattern of the 42 kDa and 55 kDa CDK9 proteins varies across different murine tissues, with liver having higher expression of the 55 kDa protein than the 42 kDa protein . The 55 kDa, but not 42 kDa, CDK9 protein has also been shown to accumulate in the nucleolus of HeLa cells .
Regulation of in CCAPs in CD4+T lymphocytes and monocytes/macrophages
The expression patterns of the protein subunits of CCAPs in CD4+ T cells and monocytes/macrophages have important implications for HIV-1 replication, as these are the two major cell types infected by HIV-1 in vivo. Resting CD4+ T cells do not support HIV-1 replication, in part because P-TEFb function is limiting [57, 58]. In resting CD4+ T cells, CCNT1 levels are low, and this involves repression of translation of CCNT1 mRNA by miRNAs . Upon T cell activation, this repression is released and there is a strong induction of CCNT1 protein levels [60–62]. In resting CD4+ T cells, the catalytic function of CDK9 is further repressed by dephosphorylation of T186 in the T-loop of the kinase; upon T cell activation there is a rapid induction of T-loop phosphorylation and CDK9 kinase activity [63, 64]. Also in resting CD4+ T cells, the level of the 7SK snRNP complex is low, and upon T cell activation there is a large increase the level of the 7SK snRNP [65, 66]. In preliminary experiments, we found that the protein subunits of the five CCAPs investigated in this study are expressed at low level in resting CD4+ T-cells and are induced upon cellular activation, suggesting that the levels of these CCAPs are up-regulated following T cell activation. Thus, it appears that resting CD4+ T cells require only very low levels of CCAPs to support basal metabolism in quiescent T cells.
Similarly, CDK9 and CCNT1 are highly regulated in monocytes and macrophages. In monocytes, miR-198 represses expression of CCNT1  and although the 42 kDa CDK9 protein is generally expressed at a high level, the CDK9 T-loop is not phosphorylated . Upon macrophage differentiation, CCNT1 levels are strongly up-regulated and the CDK9 T-loop is phosphorylated [68–70]. The 55 kDa CDK9 protein is also up-regulated during macrophage differentiation . Similar to resting and activated CD4+ T cells, the 7SK snRNP complex is expressed at only low levels in quiescent monocytes and it is induced following macrophage differentiation . Upon extended time in culture, CCNT1 expression is shut-off by proteasome-mediated proteolysis . However, CCNT1 but not CCNT2 expression can be up-regulated in late differentiated macrophage by either HIV-1 infection or activation with pathogen-associated molecular patterns (PAMPs). With the exception of Core P-TEFb and the 7SK snRNP, the expression patterns of CCAPs shown in Figure 1 have not been examined in monocytes and macrophages.
Four of the novel CCAPs identified by our analysis remain to be investigated for potential roles in Tat function (Figure 1, complexes F, H, J, and the CBP/p300, the 15 subunit INO80 complex, 20 subunit SWI/SNF complex, and 30 subunit Mediator complex). Our preliminary results with the ATR/ATRIP CCAP suggest that it may act to positively regulate Tat function and viral gene expression, in agreement with a previous study . ATR is a protein kinase involved in the DNA damage response, and it is activated by the HIV-1 Vpr protein . The finding that ATR/ATRIP is found in a complex with CDK9/CCNT1 raises the intriguing possibility of a mechanistic link between Tat and Vpr functions. Complex H contains RNAGAP1/RANBP2/RFPD3 and is involved in nuclear pore function, suggesting that Tat activation of RNAP II elongation may be coupled to RNA export and perhaps HIV-1 Rev function via this complex. In summary, our identification of 12 multi-protein complexes which contain CDK9/CCNT1 reveals considerable biological complexity in this cellular kinase and will serve as the basis for future research on molecular mechanisms involved in HIV-1 gene expression and replication.
Our results identified a very large potential complexity in the biological functions of CDK9 and CCNT1. Our results suggest that at least five newly identified multi-protein complexes contain both CDK9 and CCNT1 and may reduce the amount of these proteins available for Tat function.
Cell culture and activation
TZM-bl, HeLa, and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Jurkat CD4+ T cells and Jurkat 2D10 cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. 2D10 cells were activated using Dynabeads CD3/CD28 T cell expander beads (Invitrogen) according to the manufacturer’s protocol.
SiRNA and plasmid transfections
Control and siRNAs against individual subunits of CCAPs were purchased from Santa Cruz Biotechnology, CA as pools of three target specific siRNAs. The pool of three siRNAs was specific for distinct target sites in the transcript of the protein to be depleted. This approach has an advantage over using individual siRNAs since depletion of target protein could be achieved with lower amount of siRNA reducing the risk of off-target effects. SiRNAs (10–30 pmol) was delivered into TZM-bl, HeLa, Jurkat or 2D10 cells by either a reverse or traditional transfection method. For reverse transfections, TZM-bl or HeLa cells were seeded in 10 cm plates at 24 hours before experiment. On the day of transfection, the cells were collected by trypsinization and suspended in DMEM with 10% FBS and no antibiotics (50,000 cells/ well/sample) in 24 well tissue culture plates. Transfection was carried out using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. SiRNAs and Lipofectamine RNAiMAX were diluted in Opti-MEM1 and mixed together. The siRNA-transfection reagent mixture was incubated at room temperature (RT) for 20 minutes and added to the cells. For traditional transfections, the protocol used was as described above except that TZM-bl or HeLa cells were seeded in 24 well tissue culture plates (50,000 cells/well/sample) at 18 hours before experiments. Jurkat cells and 2D10 cells were seeded the same day of experiment and transfected with siRNAs as described above.
In experiments with TZM-bl cells in 24 well tissue culture plates, cells were transfected for 48 hours with 50 ng of pFLAG- wild type HIV-1 Tat (wtTat) or pFLAG-pro18IS-HIV-1 Tat (mTat) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In other plasmid transfection experiments carried out in HeLa cells, 500 ng HIV-1 LTR reporter plasmid and 50 ng Tat expression plasmid were transfected as described for TZM-bl cells. In these experiments, cells were also transfected with 500 ng pNF-kB-Luc and 50 ng pcDNA empty vector (pCMV parent) ensuring equal amounts of DNA were transfected.
Immunoblot and immunoprecipitation analysis
Cells were lysed with EBCD buffer (50mM Tris–HCl, pH 8.0, 120mM NaCl, 0.5% NP-40, 5mM dithiothreitol) containing protease inhibitor cocktail (Sigma). Immunoblotting was performed as described previously . TTF2, FBXO11, WRNIP1, TPR, PPP1R10, Hsp70 and β-actin were probed using antibodies against TTF2 (Abcam, 1:1000), FBXO11 (Bethyl Labs, 1:2000), WRNIP1 (Santa Cruz, 1:1000), TPR (Santa Cruz, 1: 1000), PPP1R10 (Abcam, 1: 1000), and β-actin (Sigma) (1:5000), respectively. Immunoblots were quantified using Image J software . Immunoprecipitation was carried out as described previously [65, 74].
Luciferase reporter assays
Cells were washed with sterile PBS and cell lysates prepared with Cell Culture Lysis Buffer (Promega). Cell lysates were analyzed for Luciferase activity using Luciferase Assay Kit (Promega) according to manufacturers’ protocol. Luciferase assay products were measured using a luminometer (Turner). Total protein in the lysates was estimated using a Bradford assay (Bio-Rad) and used to normalize the Luciferase readings.
Flow cytometry analysis
2D10 cells were examined for GFP expression on a BD Fortessa flow cytometer. 10,000 events were collected and the data analysed using BD FACS Diva software. For visualization of the data as a dot plot, PE was used as a placeholder fluorophore.
We thank Susan Marriott (Baylor College of Medicine) for the NF-κB- Luciferase plasmid and Jonathan Karn (Case Western Reserve University) for the Jurkat 2D10 cells. This work was supported by NIH grant AI AI089439 to APR and T32 AI7456 to RR.
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