The dynamics of cortical actin is altered in INPP5B-depleted cells. (A) Representative TIRF microscopy images at the indicated time points of INPP5B^{Degron/Degron} cells expressing LifeAct-EGFP after stimulation on immobilized anti-IgM. See Videos 5 and 6 for the complete time-lapse TIRF microscopy images. (B) Analysis of F-actin (LifeAct-EGFP) foci clearance along time in vehicle- vs. auxin- treated INPP5B^{Degron/Degron} cells. Data (n = 45 foci) from three independent experiments were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. Error bars represent mean ± SD. (C) Protein extracts from BCR-stimulated INPP5B^{Degron/Degron} cells were blotted for cofilin and ezrin phosphorylation at the indicated time points. The asterisk marks a nonspecific band. (D) Quantification of three such blots by densitometry is shown in D for cofilin (top) and ezrin (bottom). Bars represent mean ± SD, and P values were calculated as above. (E) GTP-bound Cdc42 was detected using GST-PAK1 RBD pulldown followed by blotting against Cdc42. Quantification of three such blots by densitometry is shown on the right. GTP levels of Cdc42 are expressed relative to total Ras, and data were normalized to unstimulated vehicle (control). Data were analyzed by two-way ANOVA, and P values were calculated using Sidak multiple comparisons test. Error bars indicate SEM. Source data are available for this figure: SourceData F6.