Fig. 3

AZGP1 secretion is inversely correlated with MIIP expression. a–b The concentration of AZGP1 in the supernatant of HCT116 cells a and Miip stable knockdown CMT93 or CT26.WT cells b was detected by ELISA (7 biological replicates per group). c The mRNA levels of AZGP1/Azgp1 in the indicated cells were determined by RT-qPCR (3 biological replicates per group). d Co-immunoprecipitation analysis of MIIP and AZGP1 in HCT116 cells transfected with pCMV-MIIP-Flag, by immunoprecipitation with anti-Flag and immunoblot with anti-AZGP1. e Mature adipocytes were treated with the CM from AZGP1-knockdown or NC HCT116-MIIP^+/− cells for 24 h, then washed, and the medium was changed. After an additional 24 h, protein samples were extracted and subjected to immunoblots (Representative of 3 biological replicates per group). f The concentration of free fatty acids (Left) and glycerol (Right) in supernatants of cells described in e was detected (12 biological replicates per group). g Glycosylation pattern of AZGP1 protein in HCT116 control and MIIP-overexpressing cells. Cell lysates were treated with PNGase F and subjected to immunoblot analysis (Representative of 3 biological replicates per group). h Glycosylation of AZGP1 protein in HCT116 control and MIIP-overexpressing cells. Cells were treated with Tunicamycin (1 μg/mL) or not and subjected to immunoblot analysis (Representative of 3 biological replicates per group). i HCT116 cells were co-transfected with pEX-3-AZGP1-6 × His and pCMV-MIIP-Flag (or empty vector) for 48 h, then ER and Golgi apparatus protein were extracted, followed by co-immunoprecipitation analysis. The lysates were precipitated with anti-His and immunoblotted with anti-AZGP1 and anti-MIIP. j Immunoblot analysis of the protein expression pattern of AZGP1 WT and its NQ mutants in HEK-293 cells. k LC–MS/MS-based detection of the relative N-glycosylation intensity of AZGP1 at the N259 residue in MIIP-overexpressing cells and control cells, Fold Change = 0.149 (Case/Control, 3 biological replicates per group). l HCT116 cells were transfected with the ectopic expression plasmid of AZGP1 WT and its NQ mutants for 48 h, followed by co-immunoprecipitation analysis with anti-MIIP and anti-His successively. m Co-immunoprecipitation analysis of MIIP-AZGP1-STT3A/STT3B interaction in HCT116 cells co-transfected with pCMV-MIIP-Flag and pEX-3-AZGP1-6 × His. The lysates were precipitated with anti-His and immunoblotted with anti-AZGP1, anti-MIIP, anti-STT3A, and anti-STT3B. Black spot, glycosylated AZGP1; arrowhead, non-glycosylated AZGP1. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001