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

Figure 3.

Figure 3

TRAF4 regulates the interaction of IRS-1 with IGFR and IRS-1 tyrosine phosphorylation upon IGF-1 stimulation.A, TRAF4 overexpression increased IGF-1-induced IRS-1-IGFR interaction and IRS-1 tyrosine phosphorylation. MCF-7 cells were transfected with FLAG-IRS-1 with and without TRAF4, serum starved for 24 h, and then treated with or without IGF-1 (100 ng/ml) for 15 min. Immunoprecipitation was performed using an anti-FLAG antibody followed by Western blot (left). TRAF4 and FLAG-IRS-1 overexpression and endogenous expression of indicated proteins were determined by Western blot. GAPDH was used as internal control (right). B, TRAF4 does not alter IRS-1 membrane recruitment in the presence of IGF-1. MCF-7 cells were transfected with vector control or TRAF4 expression plasmid, serum starved, and then stimulated with IGF-1 for 10 min before harvesting. Cytosol and membrane fractions were prepared using a Mem-PER plus membrane protein extraction kit. Western blot was performed to determine the distribution of IRS-1 in cytosol and membrane fraction using anti-IRS1 antibody. GAPDH was used as loading control for cytosol and whole-cell lysate fraction, whereas E-cadherin was used as loading control for the membrane fraction. C, TRAF4 does not alter the protein level and the phosphorylation level of IGF-1Rβ. MCF-7 cells were transfected with either vector control or TRAF4 plasmids. These cells were serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 5 min. Cell lysate was prepared followed by Western blot to detect phosphorylation levels of IGF-1Rβ. D, deletion of TRAF4 abolished IGF-1-induced interaction between IRS-1 and IGFR. MCF-7 cells were transfected with a pool of two different siRNAs against TRAF4 or control siRNA. MCF-7 cells were then serum starved for 24 h followed by treatment with or without IGF-1 (100 ng/ml) for 15 min. Immunoprecipitation was performed using an anti-IRS-1 antibody followed by Western blot (left). TRAF4 knockdown efficiency and endogenous expression of indicated proteins were determined by Western blot. GAPDH was used as internal control (right).