Figure 6
From: Suppression of PP2A is critical for protection of melanoma cells upon endoplasmic reticulum stress
PP2A inhibits BimEL phosphorylation through dephosphorylation of ERK in melanoma cells. (a) Knockdown of ERK1/2 blocks phosphorylation of BimEL in melanoma cells with or without undergoing ER stress. Mel-RM cells were transfected with the control siRNA or ERK1 siRNA plus ERK2 siRNA. Twenty-four hours later, cells were treated with OA (50 nM) for 24 h in the presence or absence of TM (3 μM). Thirty microgram of total protein of whole-cell lysates were then subjected to western blot analysis of Bim, pERK, ERK, and GAPDH (as a loading control). The data shown are representative of three individual western blot analyses. (b) The MEK inhibitor U0126 blocks phosphorylation of BimEL mediated by OA in melanoma cells. Thirty microgram of total protein of whole-cell lysates from Mel-RM and MM200 cells treated with OA (50 nM), U0126 (20 μM), or OA plus U0126 for 24 h were subjected to western blot analysis of Bim, pERK, ERK, and GAPDH (as a loading control). The data shown are representative of three individual experiments. (c) PP2A-C is physically associated with BimEL in MCF-7 breast cancer cells but not in melanoma cells. Whole-cell lysates from Mel-RM melanoma cells with or without treatment with TM (3 μM) for 6 h and those from MCF-7 breast cancer cells with or without treatment with TM (3 μM) for 16 h were subjected to immunoprecipitation with a mouse antibody against PP2A-C. Immunoprecipitation of whole-cell lysates from cells treated with TM with purified mouse IgG was included as a control. Thirty microgram of total protein of the resulting precipitates were subjected to SDS-PAGE and probed with an antibody against Bim and an antibody against PP2A-C. The data shown are representative of three individual experiments. (d) PP2A-C is physically associated with ERK in melanoma cells but not in MCF-7 breast cancer cells. Whole-cell lysates from Mel-RM melanoma cells and MCF-7 breast cancer cells with or without treatment with TM (3 μM) for 6 h were subjected for immunoprecipitation with a mouse antibody against PP2A-C. Thirty microgram of total protein of the resulting precipitates were subjected to SDS-PAGE and probed with an antibody against ERK and an antibody against PP2A-C. The data shown are representative of three individual experiments
