Fig. 3

HRD1 decreased protein stability of PFKP. a, b MDA-MB-231 cells were infected with Ad-GFP or Ad-HRD1 for 48 h. Immunofluorescence staining was performed for PFKP (red), HRD1 (green), and DAPI (blue). Scale bars = 10 μm. Arrows indicate the area where PFKP and HRD1 co-localized in cytoplasm. Co-immunoprecipitation assays were performed to measure the interaction between PFKP and HRD1. c Western blotting was performed to measure the level of HRD1 and PFKP proteins in each breast cancer tissue (35 cases). The relative amounts of HRD1 and PFKP protein in each tissue are expressed by the grey density of each protein per grey density of GAPDH protein. d–f The protein level, enzyme activity and mRNA level of PFKP in MDA-MB-231 cells infected with Ad-GFP or Ad-HRD1 for 48 h were measured by western blotting, PFKP enzyme activity assay and real-time PCR assays, respectively. g MDA-MB-231 cells were infected with Ad-GFP or Ad-HRD1 for 48 h. After co-culture with cycloheximide (CHX, 50 mmol/L) for 0, 3, 6, or 9 h, western blotting was performed, and the relative PFKP expression was calculated. Data are presented as mean ± SD and represent three separate experiments