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. 2016 Jun 1;291(30):15527–15539. doi: 10.1074/jbc.M116.715185

FIGURE 7.

FIGURE 7.

Requirement of Sec31 phosphorylation by CRK1 for Sec31 function. A, Western blotting to detect the overexpression (OE) of Sec31 and its phosphodeficient mutant (Sec31-7A) and phosphomimic mutant (Sec31-7D), which was induced in the presence (+) of 100 ng/ml tetracycline. Levels of TbPSA6 served as the loading control. B, effect of overexpression of Sec31 phosphodeficient and phosphomimic mutants on cell proliferation. Cells were incubated with 0.5 μg/ml tetracycline to induce the overexpression of wild-type and mutant Sec31 proteins. C, tabulation of cells with different numbers of nuclei (N) and kinetoplasts (K) of the overexpression cell lines. A total of 200 cells were counted for each time point, and error bars represent S.D. calculated from three independent experiments. D, quantification of abnormal cells in the overexpression cell lines. A total of 300 cells were counted for each time point, and error bars indicate S.D. calculated from three independent experiments. E, quantification of cells with discrete TbGRASP spots in control and overexpression cells. A total of 300 cells were counted for each cell line, and results were presented as mean percentage ± S.D. (n = 3). ***, p < 0.001. F, effect of overexpression of mutant and wild-type Sec31 on TbGRASP localization. Non-induced control and tetracycline-induced overexpression cells (Sec31 wild-type and mutants) were co-immunostained with anti-TbGRASP and FITC-conjugated anti-HA antibodies. Arrows indicate the TbGRASP signal in the Golgi. Scale bar, 5 μm. G, effect of overexpression of Sec31, Sec31-7A, and Sec31-7D on the protein level of TbGRASP, which was detected by anti-TbGRASP. TbPSA6 served as the loading control. H, effect of overexpression of mutant and wild-type Sec31 on TbGntB localization. TbGntB was endogenously tagged with a PTP epitope in cells containing the overexpression construct. Overexpression of wild-type and mutant Sec31 was induced with tetracycline, and control and overexpression cells were immunostained with anti-protein A antibody to detect TbGntB-PTP. Arrows indicate TbGntB-PTP signal in the Golgi. Scale bar, 5 μm. I, effect of overexpression of Sec31, Sec31-7A, and Sec31-7D on the protein stability of TbGntB, which was tagged with PTP and detected by anti-protein A antibody. TbPSA6 served as the loading control. DIC, differential interference contrast.