Depletion of NuMA from HeLa cells by siRNA. (A) HeLa cells were treated with control dsRNA, or two specific siRNAs against NuMA (siRNA 1 and 2; see main text for description of targeting sequences). Cells were lysed after 72 hours, and lysates were analysed by immunoblot, using antibodies against NuMA (monoclonal antibody NA09L, Calbiochem) or against alpha-tubulin (monoclonal antibody DM1a, Sigma-Aldrich). (B) Immunoblots of decreasing amounts of HeLa lysates from control cells, or cells treated with NuMA siRNA1 for 72 or 96 hours, respectively. Amounts were loaded as indicated. The blots were probed with antibodies against NuMA, or nucleolin as a loading control. Depletion efficiency was determined by scanning of the blots and comparing the intensity of NuMA from depletion experiments to the dilution series of control lysate. (C) Testing of the depletion efficiency of NuMA by immunofluorescence. Left, cells treated with control RNA for 72 hours; right, cells treated with NuMA siRNA1. Bar, 20 μm.