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. 2015 Mar 18;14(10):1568–1582. doi: 10.1080/15384101.2015.1026489

Figure 2.

Figure 2.

CHO1-pS716S717 localizes to centrosomes during mitosis. (A, B) Subcellular localizations of CHO1-pS716S717 (A) and CHO1 (B) in synchronized HeLa-S3 cells. Anti-CHO1[GS] is CHO1-specific antibody that recognizes the F-actin binding regions (FABR). (C) Peptide competition assay in which the anti-pS716S717 antibody was pre-incubated with phosphorylated (+) or non-phosphorylated (−) CHO1-S716S717 peptides prior to immunostaining. (D) Subcellular localization of CHO1-pS716S717 in metaphase large tumor suppressor (Lats)1 KO/HeLa-S3, Lats2 KO/HeLa-S3, and parental HeLa-S3 cells synchronized at mitosis by a thymidine single block-and-release (10 h). (E) Subcellular localization of metaphase CHO1-pS716S717 in HeLa-S3 cells that were transfected with siRNAs against the common 3′-UTR of CHO1 and MKLP1 and synchronized at mitosis by a thymidine single block-and-release (10 h). The GL2 siRNA was used as a negative control. The graph shows the pixel intensity of pS716 signals at the centrosome. Data represent the mean ± SD of n = 3 experiments. (F) Localizations of the pS716S717 and pS814S807 versions of CHO1 and/or MKLP1 in HeLa-S3 cells undergoing mitosis. A–F. Scale bar, 10 μm. (G) Overview of the subcellular localizations of CHO1-pS716S717 and CHO1-pS814S807/MKLP1-pS710 during the cell cycle. (H) Immunoblot analyses of exogenous CHO1-pS716S717 in HeLa-S3 cells that were transfected with 6Myc-tagged full-length MmCHO1-WT (wild type),-SA (S717A),-2SA (S805A, S807A) or-3SA (S717A, S805A, S807A), and treated with (Noc.) or without (Ctl.) nocodazole (80 ng/ml) for 18h.