Figure 2. siRNA reduces protein and mRNA levels of TβRII

A: siRNAs suppress TβRII protein expression in corneal fibroblasts. Total cell lysates from human corneal fibroblasts treated with hT1 siRNA or control, scrambled siRNA for 48 h were separated on 10% SDS polyacrylamide gels and immunoblotted with a TβRII specific antibody. Lane 1 contains lysate from cells incubated with TransIT-TKO reagent alone (no siRNA). Lane 2 contains lysate from cells treated with 100 nM scrambled siRNA. Lanes 3 and 4 contain lysates of cells treated with hT1 siRNA at a final concentration of 100 nM (lane 3) or 200 nM (lane 4). In lane 5, the TβRII antibody was preincubated with antigenic peptide before probing the normal cell lysate. The TβRII band is indicated. B: siRNA reduces levels of TβRII transcript. RNA was prepared from cells treated with scrambled siRNA or hT1 siRNA for 48 or 72 h at either 100 or 200 nM concentration. Following conversion to cDNA, the samples were analyzed by real time PCR using primers to TβRII or 18S rRNA. The bar graphs show the mean expression levels of TβRII transcript normalized to 18S RNA relative to that of scrambled siRNA treated controls. Asterisks indicate that the data are significantly different from the scrambled controls (p<0.0002, n=3, Student's t test).