Figure 2

RbAp46 participates in the inhibitory action of Ras on RECK expression in T24 and 7–4 cells. (A) The 7–4 cells, transfected with or without RbAp46-specific siRNA (55.6 nM), were treated with or without IPTG for 48 hr. RT-PCR was performed using RECK- and RbAp46-specific primer sets to measure RECK and RbAp46 mRNA levels. β-actin was used as the internal control. Ras expression was confirmed by Western blotting. (B) T24 cells were infected with Ras shRNA lentivirus for 24 hr and selected by puromycin. A scrambled shRNA expressing GFP was used as the negative control. The protein expression of Ras, RECK, RbAp46 and GFP was determined by Western blotting. NC: negative control. (C) T24 cells were infected with RbAp46 shRNA lentivirus as described above. Expression of various proteins was detected by Western blotting using specific antibodies as indicated. NC: negative control. (D) The 7–4 cells that were seeded onto 24-well plates for 24 hr were transfected with RECK promoter-luciferase reporter plasmid (pGL3-RECK) followed by IPTG treatment to induce Ras expression for the times indicated. The cell lysates were harvested and luciferase activities were measured using the luciferase assay system (upper panel). Ras protein levels in the 7–4 cells were analyzed by Western blotting (lower panel). (E) The 7–4 cells were co-transfected with 0.2 μg of pGL3-RECK or Vector (pGL3) and pcDNA-RbAp46 plasmids at various amounts (0.4-1.0 μg). Luciferase activity was measured 48 hr after transfection. * indicates statistical significance at p < 0.05 and ** indicates p < 0.001 by Student’s t test analysis. (F) The 7–4 cells were co-transfected with 0.2 μg of either pGL3 vector, pGL3-RECK, pGL3-RECK + Ras or pGL3-RECK + RbAp46 cDNA, and RbAp46-specific siRNA (55.6 nM) or a control siRNA (55.6 nM). Luciferase activity was measured 48 hr after transfection.