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. 2021 May 13;296:100739. doi: 10.1016/j.jbc.2021.100739

Figure 4.

Figure 4

TRAF4 targets K1186 and K1189 of IRS-1 for ubiquitination.A, schematic representation of IRS-1 WT and the mutants. The C-terminal last 62 amino acid sequences are shown with K1186 and K1189 in red letter. B, deletion of the C-terminal (1181–1243) region of IRS-1 abolished TRAF4-mediated ubiquitination. FLAG-IRS-1 and deletion mutant was cotransfected with TRAF4 and HA-Ub into 293T cells. The IRS-1 was immunoprecipitated using a FLAG antibody, and the ubiquitinated IRS-1 was detected using an anti-HA antibody by Western blot. C, mutation of lysine 1186 and 1189 to arginine in IRS-1 abolished TRAF4-mediated ubiquitination. FLAG-IRS-1 WT or K1186_1189R mutant was cotransfected with TRAF4 and HA-Ub into 293T cells. The IRS-1 was immunoprecipitated using a FLAG antibody, and ubiquitinated IRS-1 was detected using an anti-HA antibody by Western blot. D, IRS-1 ubiquitination site mutation does not abolish the interaction between IRS-1 and TRAF4. Top, Myc-TRAF4 interacted with FLAG-IRS-1 WT or TRAF4-ubiquitin mutant IRS-1 (IRS-1 mut) in transiently transfected 293T cells. A coimmunoprecipitation experiment was performed using an anti-FLAG antibody for immunoprecipitation, followed by Western blot. Bottom, input control.