shRNA knockdown of SRF and novel CArG-containing target genes. (A) shRNA knockdown of endogenous SRF in A7r5 rat vascular smooth muscle cells (upper panel) and human coronary artery smooth muscle cells (HCASMC, lower panel). Cells were transduced with adenovirus carrying either a short hairpin (sh) to EGFP or SRF and total cell lysates harvested at the indicated days post-transduction (dpt) for Western blotting of SRF protein levels. Note the virtual absence of detectable SRF 5 dpt in both cell types; (NC) negative control protein Tuba whose gene does not contain functional CArG boxes. (B) Linear RT-PCR results showing shSRF-mediated suppression of Cnn1, Actn1, Actr3, Dstn, Flna, and Flnc mRNA expression in A7r5 smooth muscle cells. shSRF has little effect on the negative control (NC) gene Tuba, a gene that is not SRF-dependent.
