Ser352 phosphorylation relieves CH-GAR autoinhibition. (A) S352A or S352D mutant of GAR-Tail construct (197–681; FLAG-tagged) was transiently coexpressed in HEK293T cells with GAS2L1 CH domain (GFP-tagged) for anti-FLAG immunoprecipitation. Aliquots of immunoprecipitates (50%) and cell-lysate inputs (1%) were immunoblotted with anti-FLAG and anti-GFP. IP, immunoprecipitation. Amounts of coimmunoprecipitated GFP-CH were quantified and are presented as means ± SD from three independent experiments. Unpaired Student’s t test (two-tailed); **, P < 0.001. (B) GAS2L1 constructs (Bio-GFP–tagged) were transiently expressed in HEK293T cells for streptavidin pull-downs. WT, WT GAS2L1; S352A and S352D, GAS2L1 mutants. To disrupt F-actin, cells were treated with latrunculin B (Lat B) for 30 min before harvesting. Samples of pull-downs (50%) and cell extracts (1%) were analyzed by immunoblotting. F-actin was quantified from pull-downs performed without latrunculin B treatment, and the data are presented as means ± SD from three independent experiments. One-way ANOVA; *, P < 0.01; **, P < 0.001. (C) RPE-1 gas2l1^−/− lines stably expressing GFP-GAS2L1 (WT) or mutants (S352A and S352D) were stained for Nedd1, cyclin B1, and F-actin. Boxed areas are enlarged. Centrosome-associated F-actin was quantified from G2 cells from three independent experiments, and the data are presented as means ± SD of three experimental repeats: n = 77 (WT), 79 (S352A), and 88 (S352D). One-way ANOVA; *, P < 0.01. (D) RPE-1 cells were detached for F-actin staining. G2 cells were identified through anti-cyclin B1 staining, and centrosomes were labeled with anti-Nedd1 antibody. Boxed areas are enlarged. Intensity of centrosome-associated F-actin was determined and is presented as means ± SD from three independent experiments: n = 91 (cyclin B1–positive) and 99 (cyclin B1–negative). Unpaired Student’s t test; *, P < 0.01. Scale bars, 5 µm in cell micrographs and 2.5 µm in enlarged images.