Fig. 4: CHC associates with SCYL2 and PTEN.

A Cell lysates from SU9T-01 were precipitated with anti-SCYL2 or -CHC antibodies, and the precipitated proteins were immunoblotted with the indicated antibodies. B CHC or Rab5 (Alexa Fluor-488, green) and SCYL2 (Alexa Fluor-555, red) were detected in SU9T-01 by immunofluorescent staining, and cell nuclei were stained with DAPI (blue). Scale bar, 5 µm. A region identified by the white box is further magnified to show the colocalization. C Quantification of colocalization of SCYL2 with CHC or Rab5 was carried out using Pearson’s correlation coefficient from 20 representative images. The minimal value for significant colocalization is 0.5 (dotted line). The mean and SD are shown (n = 20). D Cell lysates of 293T cells transfected with EGFP-SCYL2, Flag-PTEN, and HA-CHC were immunoprecipitated with anti-GFP or anti-Flag antibodies, and immunoprecipitates were detected by western blotting using the indicated antibodies. E Cell Lysate from SU9T-01 cells (parental, shluc, shCHC-1, and shCHC-2) was investigated using antibodies specific to each immunoblot. The results are representative of three independent experiments. Bar graphs show the quantification of relative band intensity normalized to β-actin. The mean and SD are shown (n = 3); #p < 0.05, versus parental. F Cell lysate form SU9T-01 cells (parental, shluc, and shSCYL2) was investigated using specific antibodies in immunoblots. PTEN immunoprecipitated from SU9T-01 cells were analyzed by immunoblotting with the indicated antibodies. The results are representative of three independent experiments. Bar graphs show the quantification of the relative band intensity normalized to immunoprecipitated PTEN. The mean and SD are shown (n = 3); #p < 0.05, versus parental.