FIG. 3.

Confirmation of siRNA suppression of protein expression. Where antibodies could be obtained and were of sufficient sensitivity to detect endogenous protein, Western blots were performed after treatment with siRNAs (as indicated above the blots). For each experiment, cell lysates were made from untreated cells (lane 1), cells treated with transfection reagent alone (lane 2), cells treated with transfection reagent together with nontargeting siRNA (lane 3), or transfection reagent with siRNA targeting the indicated gene (lane 4). The lysates were made 2 days after siRNA transfection in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer with 2% β-mercaptoethanol. After samples were boiled for 5 min, each was loaded on 10 or 12% polyacrylamide gel electrophoresis gels as appropriate and transferred to nitrocellulose membranes and the blots were stained with each specific antibody (upper panels). The blots were then destained and reprobed with β-actin-specific antibody which served as a gel-loading control (lower panels).