Phosphorylation at Ser352 is required for GAS2L1 function in centrosome separation. (A) Asynchronous RPE-1 cells were transfected with GFP or the following GFP-tagged GAS2L1 constructs: WT and mutants 5A, 5D, S352A, S352D, S352A/4D, and S352D/4A. Centrosomes were labeled with anti-γ-tubulin antibody. Centrosome distance was measured in the transfected cells from three independent experiments: n = 96 (GFP), 110 (WT), 120 (5A), 114 (5D), 112 (S352A), 110 (S352D), 100 (S352A/4D), and 100 (S352D/4A). Arrowheads: centrosomes. (B) GAS2L1 expression was examined in RPE-1 parental cells and sublines. gas2l1 KO, gas2l1-knockout line; GFP-GAS2L1 (WT) or mutants (S352A and S352D) were stably expressed in the knockout line. Anti-GAS2L1, anti-GFP, and anti-α-tubulin immunoblotting was performed. (C) RPE-1 parental cells and sublines were stained for CENP-F and γ-tubulin. Centrosome distance during late G2 was measured from CENP-F–positive cells in three independent experiments: n = 161 (parental), 170 (gas2l1 KO), 150 (WT), 162 (S352A), and 149 (S352D). Arrowheads: centrosomes. (D) RPE-1 lines as in C were arrested with aphidicolin and then released and treated with STLC. Centrosomes were labeled through anti-centrin staining. Centrosome distance was measured in mitotic cells (arrested in prometaphase by STLC) from three independent experiments: n = 100 (parental), 100 (gas2l1 KO), 120 (WT), 123 (S352A), and 121 (S352D). (A, C, and D) Arrowheads, centrosomes. Scale bars, 10 µm in cell micrographs and 5 µm in enlarged images. χ² test; **, P < 0.001; ***, P < 0.0001; n.s., not significant.