Retrograde movement of dynein complexes containing IC-2C in siRNA-treated PC12 cell neurites. (A) Knockdown of endogenous ICs but not GFP–IC by siRNA. The GFP–IC-2C stable PC12 cell line was transfected with either siRNA oligonucleotides to the 3′ untranslated region of the rat IC-2 gene (IC-2 siRNA) or control siRNA (Sc siRNA) by electroporation. The cells were grown for 72 h after transfection, and cell lysates were analyzed by SDS-PAGE and Western blotting. The blot was probed with the pan IC antibody (74.1) to identify the endogenous ICs (IC) and the GFP–IC-2C (GFP–IC), and, for a loading control, a tubulin antibody (Tubulin). (B) Retrograde dynein movement in siRNA-treated PC12 cell neurites. IC-2 siRNA-transfected PC12 cells with stable expression of GFP–IC-2C were grown on coverslips and differentiated by the addition of NGF. 72 h after transfection, the dynein motility was imaged. The panels show a video of a portion of a PC12 cell neurite (Video 2). The arrows indicate one puncta with retrograde movement between the two frames. The time between the video panels is indicated on the right. Although not identified with arrows, many other puncta were moving in the anterograde and retrograde directions. Bar, 5 μm.