Fig. 9.

Role of TRAF6 and Akt2 in LPS-induced Akt ubiquitination. Cardiomyocytes from WT and Akt2 knockout mice were challenge with LPS (1 µg/ml) for 4 hrs in the absence or presence of the TRAF6 peptide inhibitor or control peptide (300 µM) prior to immunoblotting analysis. A: Cardiomyocyte samples from WT and Akt2^−/− mice were immunoprecipitated with the anti-pan Akt antibody. Akt1 and ubiquitin levels were detected using western blotting. Pan Akt was used as the input and ubiquitin was pulled down with pan Akt and detected by western blotting. Akt1 level was also examined in the protein extract pulled down by pan Akt. AminoLink Plus coupling resin uncoupled with the pan Akt antibody was employed as the negative control. B: Quantitative analysis of Akt1 levels in protein extracts pulled down by pan Akt; C: Quantitative analysis of ubiquitinated protein (Ub) pulled down with pan Akt; D: Cardiomyocyte samples from WT and Akt2^−/− mice were immunoprecipitated with the anti-ubiquitin antibody. Ubiquitin was employed as the input; Akt2 and Akt1 protein levels were detected using western blotting. AminoLink Plus coupling resin uncoupled with the ubiquitin antibody was used as the negative control; and E: Quantitative analysis of Akt2 protein precipitated with ubiquitin. Mean ± SEM, n = 4–5 mice per group, * p < 0.05 vs. WT group, # p < 0.05 vs. WT-LPS group.