Fig. 7

Trans-regulator activity of P50 on MLV promotor expression. (A) MLV promotor expression was monitored with luciferase assays. The NIH 3T3 cells were co-transfected with 1 µg of bidir MLV-LTR plasmid and increasing amounts of p57cDNASD’ construct (1.5 to 0.01 µg) expressing untagged P50. Luciferase activity was analyzed 48 h post-transfection and normalized to protein concentration, measured by the Bradford assay using BSA as a standard. The activity observed with the MLV reporter alone was used as a reference (100%). Experiments were performed in triplicates. Average luciferase activities (arbitrary units) for sense and antisense were 171,639 and 4122, respectively. Values were compared using unpaired Student’s t-test (ns: not significant, p ≤ 0.1 and ****p ≤ 0.0001). (B) NIH 3T3 cells were cotransfected with plasmids expressing the mutant F1-MLV and p50-GFP and 4 8 h post transfection, Gag or P50-GFP expressions were monitored in cells and supernatants by WB analysis and (C) cellular MLV FL RNA was quantitated by RT-qPCR. (D) Chromatin immunoprecipitation of P50. NIH 3T3 cells were co-transfected with plasmids expressing F1 and P50-GFP or GFP alone. Two days post transfection cell extracts were collected and used for a ChIP experiment with an anti-GFP antibody. DNA fragments co-precipitated with P50-GFP were analyzed by PCR using probes recognizing MLV promotor. The plasmid encoded MLV was used as PCR positive control (+) and cell extract from untransfected cells was used as PCR negative control (-)