Non-phosphorylatable mutant of SGT1 rescues mitotic defects.
A, cells expressing PHLPP1 shRNA were transfected with a phospho-dead SGT1 4A mutant or phospho-mimetic SGT1 4E mutant. Localization of HEC1 to kinetochores in these cells was tested by immunofluorescence (scale bar, 2 μm). Quantification of cells with defective HEC1 localization is shown on right (n = 50 cells each). **, p < 0.01, Student's t test. B, localization of CENP-E to kinetochores was also tested in these cells by immunofluorescence (scale bar, 2 μm). Quantification of cells with defective CENP-E localization is shown on right (n = 50 cells each). ***, p < 0.001, Student's t test. C, cells expressing PHLPP1 shRNA were transfected with a phospho-dead SGT1 4A mutant and then processed for immunofluorescence staining with α-tubulin antibody to check the spindle defects. D, γ-tubulin antibody for centrosome defects (scale bar, 2 μm). Quantification of the data is shown on right (n = 50 cells each). *, p < 0.05; **, p < 0.01, Student's t test.