Displacement of endogenous BRCA1. (A) CLSM of PMA-stimulated CHO–wild type (CHO-WT) and CHO cells stably expressing high levels of EGFP-CTD (CHO-High) labeled with TRITC-phalloidin and anti-Ser988 BRCA1 antibody (Alexa Fluor 647). Pronounced PM colocalization of endogenous BRCA1 and F-actin was observed in CHO-WT (left). In CHO-High, PM colocalization of EGFP-CTD and F-actin was observed, whereas endogenous BRCA1 was absent (right). The endogenous BRCA1 distribution in the cytoplasm remained unchanged. Bars, 5 µm. (B) Mean intensity z projection of representative TRITC-phalloidin– and anti-Ser988 BRCA1 antibody (Alexa Fluor 647)–labeled CHO-WT and CHO-High z stacks. PM colocalization of endogenous BRCA1 and F-actin was prominent in CHO-WT cells but clearly reduced in CHO-High cells. The marked white outlines indicate the selected area for calculation of colocalization parameters. (C) Quantitative displacement graph showing the distribution of all colocalization percentage values of Ser988 BRCA1 over F-actin from each plane of seven individual z stacks per CHO cell type. Overlap of Ser988 BRCA1 with F-actin is higher in CHO-WT than in CHO-High: CHO-WT, mean = 74.97 ± SEM = 1.49 (n = 57); CHO-High, mean = 61.17 ± SEM = 2.791 (n = 48). Horizontal lines indicate means. Nonparametric two-tailed Mann–Whitney test, P < 0.001; unpaired two-tailed t test with Welch’s correction, P < 0.001.