Fig 1: Loss-of-function approaches for modulating cofilin activation and cofilin-mediated actin severing. (A) To complement siRNA-mediated depletion of cofilin, cofilin activation was enhanced by inhibiting LIMK, which phosphorylates cofilin on S3. The ability of cofilin to sever actin filaments was modulated by depleting either Wdr1 or Cotl1. (B) A20 B cells were transfected with control non-targeting siRNA, cofilin siRNA, Wdr1 siRNA, or Cotl1 siRNA. Cell extracts were analyzed by immunoblotting. Loading controls were actin or the endogenous Ig light (L) chain of the A20 B cells. Representative blots are shown. (C–E) In C, A20 B cells were pre-treated with DMSO or 50 µM LIMKi3 for 1 h before being stimulated with 20 µg/mL anti-IgG for the indicated times. In (D), primary murine B cells were pre-treated with DMSO or 1 µM LIMKi3 for 1 h before being stimulated with 20 µg/mL anti-IgM. In (E), A20 B cells that had been transfected with control siRNA or Wdr1 siRNA were stimulated with 20 µg/mL anti-IgG. Representative p-cofilin and total cofilin immunoblots are shown (left panels). The p-cofilin/total cofilin ratios were normalized to the ratio in unstimulated (0 min) control cells (=1.0). The means ± SEM from three independent experiments are graphed for each time point (right panels).