Figure 4

Co-immunoprecipitation of spliced or unspliced MLV RNA with TapΔC factor. NIH3T3 cells were cotransfected with 10 μg of peGFP-TAPΔC and 10 μg of pMov9.1 or 7 μg of peGFP-TAPΔC and 10,5 μg of p3-CCCC (4-CTE). At 24 h p.t., cells were extracted with 1 ml of lysis buffer (1% triton X-100, 150 mM NaCl, 50 mM Tris–HCl, complete anti-protease cocktail from Roche) and precleared with Protein A sepharose beads (invitrogen) during one hour at 4°C. 100 μl of cell lysates were saved to control transfection efficiencies by monitoring RNA levels (input). 900 μl of cell lysates were incubated 3 hours at 4°C with beads that specifically bind GFP (GFP-Trap® Chromotek) in order to co-immunoprecipitate GFP-TapΔC/RNA complexes. After disruption of the RNP complexes, RT-PCRs were performed on the precipitated RNA and the input to specifically amplify MLV RNA, or 4-CTE RNA, and two cellular RNA controls: actinB mRNA and U6 snRNA, the nuclear export of which rely on the Tap and CRM1 pathways, respectively. Control PCR experiments were systematically performed without prior RT reaction as a control for DNA contamination of RNA extracts. Amplicons were analyzed on 1% agarose gel.