Western blot analysis and immunoprecipitation were carried out as described previously73 (link)–75 . Total proteins were prepared from the tissues and cells with RIPA buffer containing protease and phosphatase inhibitors. The lysates were subjected to western blotting and the protein was separated with SDS-PAGE and transferred to PVDF transfer membranes (Millipore, Bedford, MA, USA). Corresponding antibodies were used to detect proteins. The protein bands were measured by ChemiDocTM Touch Imaging System (Bio-Rad, Richmond, CA, USA). All experiments were repeated at least three times and amount of phosphorylated protein was quantified by Image J and normalized to amount of total protein.
For immunoprecipitation and ubiquitination, HEK293T and adipocytes were homogenized in lysis buffer. 1 mg of cell lysate protein was immunoprecipitated with indicated antibodies at 4 °C overnight. Immunoprecipitations were pulled down with Protein A/G Agarose followed by western blot analysis. For ubiquitination assays, cells were lysed with lysis buffer plus 2 mM N-Ethylmaleimide (NEM) and denudated by heating for 10 min, and centrifuged at 14,000 g for 15 min at 4 °C. The subsequent steps for the in vivo ubiquitination assay were carried out as described above in methods of immunoprecipitation and western blotting experiments.
The following antibodies were used: anti-TRPM7 (Sigma-Aldrich, AB15562), WB 1:500; anti-phospho-IRS-1 (Ser307) (Cell Signaling Technology, 2381), WB 1:1000; anti-IRS-1 (Cell Signaling Technology, 2382), WB 1:1000; anti-phospho-Insulin Receptor β (Tyr1345) (Cell Signaling Technology, 3026), WB 1:1000; anti-Insulin Receptor β (Cell Signaling Technology, 3025), WB 1:1000; anti-phospho-Akt (Ser473) (Cell Signaling Technology, 4060), WB 1:1000; anti-Akt (Cell Signaling Technology, 9272), WB 1:1000; anti-phospho-NFκB-p65 (Ser536) (Cell Signaling Technology, 3033), WB 1:1000; anti-NFκB-p65 (Cell Signaling Technology, 8242),WB 1:1000; anti-phospho-IKKβ (Ser180) (Cell Signaling Technology, 2694), WB 1:1000; anti-IKKβ (Cell Signaling Technology, 8943),WB 1:1000; anti-IκBα (Cell Signaling Technology, 9242), WB 1:1000; anti-phospho-TAK1 (Ser412) (Cell Signaling Technology, 9339), WB 1:1000; anti-TAK1 (Cell Signaling Technology, 5206), WB 1:1000, IP 1:50; anti-phospho-CaMKII (Thr286) (Cell Signaling Technology, 12716) WB 1:1000; anti-CaMKII (Cell Signaling Technology, 50049) WB 1:1000; anti-Ub (Cell Signaling Technology, 3936), WB 1:1000; anti-Flag (Sigma, F1804), WB 1:1000, IP 10 μl for 1 mg protein; anti-HA (Sigma, H3663), WB 1:1000; anti-TRAF6 (Santa Cruz, sc-8409), WB 1:1000, IP 4 μg for 1 mg protein; anti-c-Cbl (Santa Cruz, sc-1651), WB:1:1000; anti-GAPHD (Proteintech, 60004-1-Ig), WB 1:2000; anti-α-tubulin (Proteintech, 11224-1-AP), WB 1:2000; anti-rabbit (Cell Signaling Technology, 7074), WB 1:2000; anti-mouse (Cell Signaling Technology, 7076), WB 1:2000.
For immunoprecipitation and ubiquitination, HEK293T and adipocytes were homogenized in lysis buffer. 1 mg of cell lysate protein was immunoprecipitated with indicated antibodies at 4 °C overnight. Immunoprecipitations were pulled down with Protein A/G Agarose followed by western blot analysis. For ubiquitination assays, cells were lysed with lysis buffer plus 2 mM N-Ethylmaleimide (NEM) and denudated by heating for 10 min, and centrifuged at 14,000 g for 15 min at 4 °C. The subsequent steps for the in vivo ubiquitination assay were carried out as described above in methods of immunoprecipitation and western blotting experiments.
The following antibodies were used: anti-TRPM7 (Sigma-Aldrich, AB15562), WB 1:500; anti-phospho-IRS-1 (Ser307) (Cell Signaling Technology, 2381), WB 1:1000; anti-IRS-1 (Cell Signaling Technology, 2382), WB 1:1000; anti-phospho-Insulin Receptor β (Tyr1345) (Cell Signaling Technology, 3026), WB 1:1000; anti-Insulin Receptor β (Cell Signaling Technology, 3025), WB 1:1000; anti-phospho-Akt (Ser473) (Cell Signaling Technology, 4060), WB 1:1000; anti-Akt (Cell Signaling Technology, 9272), WB 1:1000; anti-phospho-NFκB-p65 (Ser536) (Cell Signaling Technology, 3033), WB 1:1000; anti-NFκB-p65 (Cell Signaling Technology, 8242),WB 1:1000; anti-phospho-IKKβ (Ser180) (Cell Signaling Technology, 2694), WB 1:1000; anti-IKKβ (Cell Signaling Technology, 8943),WB 1:1000; anti-IκBα (Cell Signaling Technology, 9242), WB 1:1000; anti-phospho-TAK1 (Ser412) (Cell Signaling Technology, 9339), WB 1:1000; anti-TAK1 (Cell Signaling Technology, 5206), WB 1:1000, IP 1:50; anti-phospho-CaMKII (Thr286) (Cell Signaling Technology, 12716) WB 1:1000; anti-CaMKII (Cell Signaling Technology, 50049) WB 1:1000; anti-Ub (Cell Signaling Technology, 3936), WB 1:1000; anti-Flag (Sigma, F1804), WB 1:1000, IP 10 μl for 1 mg protein; anti-HA (Sigma, H3663), WB 1:1000; anti-TRAF6 (Santa Cruz, sc-8409), WB 1:1000, IP 4 μg for 1 mg protein; anti-c-Cbl (Santa Cruz, sc-1651), WB:1:1000; anti-GAPHD (Proteintech, 60004-1-Ig), WB 1:2000; anti-α-tubulin (Proteintech, 11224-1-AP), WB 1:2000; anti-rabbit (Cell Signaling Technology, 7074), WB 1:2000; anti-mouse (Cell Signaling Technology, 7076), WB 1:2000.
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