Figure 4: Inhibitory effect of soy-glycinin-derived peptides on Cbl-b-mediated IRS-1 ubiquitination. (a) Purified soy protein isolate (SPI), lipoprotein (LP), soy glycinin (G), and soy β-conglycinin (C) were digested with trypsin, then 20 μg/mL of each of the hydrolysates was subjected to cell-free ubiquitination assay to elucidate their inhibitory effects on Cbl-b-mediated IRS-1 ubiquitination. (b) HEK293 cells transfected with mock vector/pCEFL-Cbl-b-HA, pcDNA3.1-rat IRS-1-V5, and pcDNA3-FLAG-Ubiquitin were treated with the indicated concentration of casein-derived (control) or soy-glycinin-derived peptides for 2 hours in the presence of 100 nM epoxomicin and 10 ng/mL IGF-1. Cell lysates from these cells were immunoprecipitated with an anti-V5 antibody. The immunoprecipitates were subjected to immunoblot (IB) analysis for the indicated proteins. MMSTD: molecular mass standards. Representative findings of three experiments with matching results.