Arp3- and Arp3B-containing complexes have the same actin nucleating activity. (A) In vitro pyrene actin assays using 12.5 nM of the indicated recombinant Arp2/3 complexes reveal Arp3 and Arp3B containing complexes nucleate actin equally efficient, regardless of the ARPC1A/ARPC5 and ARPC1B/ARPC5L background. The error bars represent SEM from n = 2 independent experiments. (B) The top row shows representative in vitro TIRF microscope images of branched actin formation using 2.5-nM Arp2/3 complexes containing Arp3B, ARPC1A, and ARPC5 at the indicated times (see Video 1). n = 2 independent experiments. Scale bar = 15 µm. The bottom row shows the same panels after automatic detection of filament branches (yellow nodes) and ends (red nodes) with AnaMorf ImageJ plugin. (C) Quantification of mean branch number and filament length for the indicated Arp2/3 complexes from one of the two independent TIRF experiments. Each data point represents the average of 14 and 15 actin “structures” for Arp3B-C1B-C5L and Arp3-C1B-C5L, respectively, although there are fewer structures to analyze at the beginning of the experiment than at the end.