Arp3 and Arp3B knockdown and rescue. (A) RT-PCR analysis showing the level of Arp3 (blue) and Arp3B (red) mRNA relative to control in HeLa cells after knockdown with the individual siRNA from the siGenome pools against Arp3 and Arp3B. Error bars represent SEM from two independent experiments. (B) Quantification of actin tail lengths and immunoblot analysis of the level of Arp3 in HeLa cells treated with the indicated siRNA. The error bars represent SEM from n = 2 independent experiments in which the length of 100 tails was analyzed per condition. Tukey’s multiple comparisons test was used to determine statistical significance; ****, P < 0.0001; **, P < 0.01. (C) Immunoblot analysis showing that stable expression of RNAi-resistant GFP-tagged Arp3 (blue) and Arp3B (red) restore control levels of all Arp2/3 subunits in HeLa cells treated with Arp3 siRNA. (D) Coomassie-stained gel of recombinant isoform-specific Arp2/3 complexes and corresponding immunoblot. (E) RT-PCR analysis of the level of Arp3B mRNA in HeLa cells stably expressing Cherry-GFP^PA-β-actin and treated with Arp3B siRNA. Error bars represent SEM from three independent experiments. (F) Immunoblot analysis of lysates from HeLa cells stably expressing GFP^PA-tagged Arp3 (blue) and Arp3B (red).