Ab52477

To study the effect of chelerythrine and protopine on insulin signaling we used 293T cells (ATCC) and cultured in DMEM containing 10% FBS. For insulin signaling, cells were serum starved for 16 h and then treated with chelerythrine (2 µM) or protopine (2 µM) for 1 h. Following which, cells were treated with 25 nM insulin for various times (0, 5, 10, 20 min) at 37 °C. Cells were washed with PBS and lysed in de-gassed RIPA lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 0.25% Deoxychloate, 10% Glycerol, 25 mM NaF, 10 mM MgCl₂, 1 mM EDTA, 1% Triton X-100, 0.5 mM PMSF, 10 mM Benzamidine, cOmplete protease inhibitor cocktail (Roche)). Soluble proteins were harvested by centrifugation at 13,000×g for 10 min at 4 °C and quantitated. Total proteins in the cell lysates were separated by SDS-PAGE and tyrosyl phosphorylation of the IR-β subunit and downstream AKT activation was monitored. To test the effect of suppressing H₂O₂ levels on the function of the compounds, catalase was ectopically expressed in cells for 24 h. Following which, the cells were treated with the compounds as mentioned above. Phosphorylation of the AKT activation loop at residue threonine 308 (T308) in response to insulin stimulation was observed with the phospho-specific antibody. Catalase expression was detected in the cell samples with a rabbit polyclonal antibody (Abcam, ab52477).