Figure 5: BI-1 regulates the formation of disulfide bonds during oxidative protein folding.

(a) BI-1 WT and BI-1 KO mice (8 weeks old, n = 5) were fed with HFD for 1- and 8-weeks. Liver lysates were analyzed for the presence of PDI in high molecular weight complexes (HMWCs), using non-reducing (top and 2^nd panels) and reducing (3^rd and 4^th panels) gels as described in Experimental Procedures. (b) From the 1- and 8-weeks-HFD-fed BI-1 WT and BI-1 KO mice, livers were isolated. The liver lysates were derivatized with DNPH, and PDI was immunoprecipitated from the derivatized lysates. Immunoprecipitated PDI was run on a non-reducing gel and analyzed by western blotting using anti-DNP antibody from an OxyBlot kit. PDI, which persisted in HMWCs (unresolved, long-lasting complexes between PDI and its client proteins), was found to be excessively carbonylated. Note that the expression of the main 57-kDa band, PDI, is weaker in the immunoprecitation from HFD-fed BI-1 KO mice than that from HFD-fed BI-1 WT mice. (c) Liver lysates were immunoprecipitated with anti-PDI or anti-ApoB antibody and immunoblotted with anti-ApoB or anti-PDI antibody, respectively. NFD; normal-fat diet, HFD; high-fat diet, WT; wild-type, KO; knock-out, PDI; protein disulfide isomerase, DNPH; 2, 4-dinitrophenylhydrazine, DNP; dinitrophenol.