Fig. 6

GBCDRlnc1 directly interacts with PGK1 and upregulated its protein level via inhibiting PGK1 ubiquitination in gallbladder cancer cells in vitro. a Silver-stained SDS-PAGE gel of proteins immunoprecipitated from NOZ/Dox cell extract by the sense and antisense RNA of GBCDRlnc1. The two lanes were used for mass spectrum determination by the liquid chromatography dual mass spectrometry method. The frame indicates PGK1. b RNA pull-down assay was conducted using biotin-labeled GBCDRlnc1 probe and determined the PGK1 expression by western blot assay. Antisense of the GBCDRlnc1 probe was used as negative control. c Amount of GBCDRlnc1 bound to SNRNP70 (a positive control), PGK1 or IgG (a negative control) was detected by qRT-PCR after RIP in NOZ/Dox cells. d Relative expression of GBCDRlnc1 in cell cytoplasm or nucleus of NOZ/Dox cells was determined by qRT-PCR. e The protein levels of PGK1 in Dox-resistant gallbladder cancer cells under different transfection were determined by western blot assay. f The protein levels of PGK1 in NOZ/Dox cells under different transfection with CHX (20 mg/ml) were determined by western blot assay. g The protein levels of PGK1 in NOZ/Dox cells under different transfection with MG-132 (5 μM) were determined by western blot assay. h NOZ/Dox cells under different transfection were treated with MG-132 (5 μM) for 24 h. Cell lysates were immunoprecipitated with antibodies against PGK1 or IgG. The levels of ubiquitination were analysed by western blot. Bottom, input from cell lysates. The mean ± SD of triplicate experiments were plotted, ***P < 0.001