FIG. 3.

Expression levels of RBM4 correlate with the inclusion of skeletal muscle-specific terminal exons of α-TM. (A) Total RNAs from HEK 293 cells that stably expressed FLAG-tagged RBM4 (+) or from mock cells (-) were analyzed by RT-PCR using primers (Table 1) specific to three α-TM isoforms (primer set 4 for sk, 2 for sm, and 5 for 9a′) and GAPDH (primer set 16). The PCR products were subjected to hybridization using a ³²P-labeled forward primer as probe. Western blotting was performed using anti-RBM4. The right panel shows quantitative RT-PCR analysis of the α-TM sk isoform from three independent experiments. The level of the sk mRNA was normalized to that of GAPDH in individual experiments. The bar scale with standard deviation shows activation (n-fold) of sk isoform expression upon ectopic expression of RBM4. (B) HEK 293 cells were infected with recombinant adenovirus expressing an shRNA targeting RBM4a or with mock adenovirus at the MOIs indicated above the gel panels. RT-PCR and Western blotting were performed as in panel A. Quantitative RT-PCR shows relative levels of the α-TM sk isoform in individual infections with the shRNA-expressing adenovirus compared with the mock infection; average values with standard deviations were obtained from three independent experiments.