Fig. 4.

GLCC1 interacts with HSP90 and regulate the stability of c-Myc. a Schematic design of using SILAC-based quantitative proteomic approach to identify the GLCC1-specific interactors. b Proteome-wide accurate quantification and significance. The logarithm of normalized protein ratios (H/L ratio) are plotted against protein intensities. The filled green circles represent the sense RNA-specific interactors (p < 0.05, 74 proteins, H/L ratio > 1.46), the red marked one is the validated sense-RNA interactor HSP90. The filled red diamonds represent the anti-sense RNA-specific-binding partners ((p < 0.05, 98 proteins, H/L ratio <0.66). As the anti-sense RNA is an “artificial” RNA, these proteins can be considered as non-specific binding. The blue crosses represent all the non-specific-binding proteins (p > 0.05, 1964 proteins). c Western blot of the proteins from anti-sense GLCC1 and GLCC1 pull-down assays; d Western blot of HSP90 in samples pulled down by full-length (F2) or truncated GLCC1 (F3: 1–500, F4: 1–250, F5: 1251–650); e RNA immunoprecipitation experiments were performed using anti-HSP90 antibody, and specific primers were used to detect GLCC1. f Western blot of the proteins from control and GLCC1 siRNA transfection DLD-1 cells. g Western blot of the proteins from control and GLCC1 overexpression plasmid SW480 cells. h Co-immunoprecipitation detected the interaction of HSP90 and c-Myc in the DLD-1 cells. i Immunoprecipitation assay was performed to detect the interaction between HSP90 and c-Myc after transfection of GLCC1 siRNA. j Western blot of c-Myc (PT56) expression in control or GLCC1 overexpression sample in SW480 and LoVo cells. k Western blot of c-Myc (PT56) expression in control or GLCC1 siRNA sample in DLD-1 and HT29 cells. l Western blot of the total ubiquitination proteins and c-Myc from control and GLCC1 siRNA1/2 transfection DLD-1 cells. m, n Western blot of c-Myc from control and GLCC1 siRNA1/2 transfection samples treated with DMSO or ubiquitination inhibitor MG132 in DLD-1 and HT29 cells