Fig. 7

A physiological role for C60 and C77. a, b TIRF imaging of adipocytes overexpressing TagRFPt-Akt2-WT treated with a BCNU/AF or H₂O₂ (100 µM) or b glucose oxidase (GOD) as indicated prior to stimulation with 1 nM insulin. Data are mean ± SEM. c The simplified mechanistic AKT oxidation-phosphorylation network model. Ins Insulin, IR Insulin receptor, NOX NADPH oxidase 4, DPI diphenylene iodinium, ROS reactive oxygen species, Akt-ox oxidized Akt, Akt-red reduced Akt, Akt-ox-PM oxidized PM Akt, Akt-red-PM reduced PM Akt. Reaction numbers rx (x = 0–37) denote the model reactions (Supplementary Tables 1 and 2). d Simulated response of Akt-PM and phospho-Akt levels with either insulin stimulation alone, or in combination with BCNU/AF, using a representative best-fitted model parameter set. e Model prediction of the influence of BCNU/AF on Akt recruitment in response to increasing amounts of PIP3. Predictions are averaged using three independent best-fitted parameter sets (Supplementary Table 2); Data are mean ± SEM. f Model prediction of the effect of DPI on Akt recruitment under a low (at 0 min time-point) or high level (at 10 min time-point) of insulin stimulation. Data are mean ± SEM. g Model prediction of the effect of increasing DPI concentration on Akt (left panel) and Akt substrate phosphorylation (right panel). DPI was added for 30 min prior to a 10 min insulin stimulus. h, i Experimental validation of model predictions. h Adipocytes overexpressing TagRFP-T-Akt2-WT were imaged by TIRFm and treated with either DMSO or 10 μM DPI 10 min prior to insulin stimulation. i DPI dose response. Adipocytes were pretreated with the indicated dose of DPI for 30 min prior to stimulation with 1 nM insulin. j Differential cysteine alkylation and mass spectrometry method of measuring the redox state of Akt1 cysteines. Unpaired cysteine thiols were alkylated with 12C-IPA and the oxidized thiols with 13C-IPA following reduction with DTT. k Box (inter-quartile range; median) and whiskers (min:max) plots showing the oxidation of Cys60 and Cys297 in NIH/3T3 fibroblasts in the presence or absence of 10 nM insulin. Peptide were binned into 0–5 min and 10–60 min insulin periods. Two biological replicates were performed. *p < 0.05 by two-sided Wilcoxon rank sum test.