Figure 3.

Inhibitory effect of Cblin-like synthetic peptide on Cbl-b-mediated degradation and ubiquitination of IRS-1. HEK293 cells were transfected with mock vector/pCEFL-Cbl-b-HA, pcDNA3.1-rat IRS-1-V5, and pcDNA3-FLAG-Ubiquitin and then treated with 100 nM epoxomicin at 2 hours before treatment with each peptide at the indicated concentrations. One hour later, these cells were treated with 10 ng/mL IGF-1 for 1 hour. Immunoprecipitates (IP) from whole cell lysates incubated with an anti-IRS-1 antibody were subjected to immunoblot (IB) analysis for the indicated proteins. The densitometric measurement of FLAG-Ub (the area indicated by right parenthesis) was performed with an ImageJ software (National Institute for Health). MMSTD: molecular mass standards. Representative findings of three experiments with matching results.