FIG. 13.

Analysis of the MDM-p53-Bak, GSK-3β-MCL-1, and FOXO3A-Bim pathways in mock and Akt1/2 knockdown cells. (A) Oxidative stress differentially regulates MDM2 phosphorylation in the mock and Akt1/2 shRNA plasmid-transfected HLECs. Both mock and Akt1/2 shRNA plasmid-transfected HLECs were either mocked treated (by H₂O) or treated with 50 μM H₂O₂ for 2 h. After treatment, the cells were harvested for analysis of total MDM2 and MDM2 phosphorylation at S166, total p53 and p53 phosphorylation at Ser-15, and β-actin (as a loading reference) using the method as described in the Materials and Methods section. Note that in Akt1/2 knockdown cells, MDM2 phosphorylation at S166 was significantly downregulated. (B) Oxidative stress differentially regulates GSK-3β phosphorylation in the mock and Akt1/2 shRNA plasmid-transfected HLECs. Both mock and Akt1/2 shRNA plasmid-transfected HLECs were either mocked treated (by H₂O) or treated with 50 μM H₂O₂ for 2 h. After treatment, the cells were harvested for analysis of total GSK-3β and GSK-3β phosphorylation at S9, MCL-1 level and β-actin (as a loading reference) using the method as described in the Materials and Methods section. Note that in Akt1/2 knockdown cells, GSK-3β phosphorylation at S9 was significantly downregulated. (C) Oxidative stress differentially regulates FOXO3A phosphorylation in the mock and Akt1/2 shRNA plasmid-transfected HLECs. Both mock and Akt1/2 shRNA plasmid-transfected HLECs were either mocked treated (by H₂O) or treated with 50 μM H₂O₂ for 2 h. After treatment, the cells were harvested for analysis of total FOXO3A and FOXO3A phosphorylation at T32, Bim level and β-actin (as a loading reference) using the method as described in the Materials and Methods section. Note that in Akt1/2 knockdown cells, FOXO3A phosphorylation at T32 was significantly downregulated.