Fig. 3: ch-TOG promotes incorporation of γTuRC at PCM and subdistal appendages.

a U2OS cells transfected with control or ch-TOG siRNA #1 were fixed and stained with antibodies against the indicated proteins. b Intensities of centriolar γ-tubulin, NEDD1, and PCNT staining in cells as in (a) were quantified, normalized to the average of the respective controls, and plotted. γ-tubulin staining: N = 3 independent experiments; total number of cells analyzed, 75 (Control RNAi) and 69 (ch-TOG RNAi); ***p = 0.0004. NEDD1 staining: N = 3 independent experiments; total number of cells analyzed, 312 (Control RNAi) and 327 (ch-TOG RNAi); **p = 0.0042. PCNT staining: N = 2 independent experiments; total number of cells analyzed, 151 (Control RNAi) and 129 (ch-TOG RNAi); p = 0.3865 (ns, not significant). The horizontal bars and whiskers indicate median and interquartile range, respectively, of the plotted data points. Statistical significance was determined by unpaired, two-tailed t test with Welch’s correction without multiple comparison. c U2OS cells transfected with control or ch-TOG siRNA #1 were co-stained with antibodies against the indicated proteins. Performed twice with similar result. d U2OS cells treated with control or NEDD1 siRNA, with or without microtubules, were co-stained with antibodies against ch-TOG and γ-tubulin. e Centriolar ch-TOG intensities were quantified as in (d), normalized to the average of the intensities of the control, and plotted. N = 2 experiments, total number of cells analyzed, 30 per condition, **p = 0.0015. The horizontal bars and whiskers indicate median and interquartile range, respectively, of the plotted data points. Statistical significance was determined by unpaired, two-tailed t test with Welch’s correction. Illustrations indicate centriole orientations in the respective images. Scale bars, 1 μm. f, g Lysates from HEK293T cells transiently expressing Flag-BirA or Flag-BirA-GCP3 and grown in the presence of biotin for 24 h were subjected to affinity pulldowns using anti-FLAG antibody and streptavidin-coupled beads. The pulldown precipitates were subjected to immunoblot and probed with anti-ch-TOG, anti-γ-Tubulin, anti-FLAG, and anti-GAPDH antibodies. Detection of GAPDH was used as control. The results were replicated in two independent experiments. Source data are provided as a Source Data file.