Figure 7. Superoxide mediates dephosphorylation of Bcl-2 and ERK1/2 induced by intense heat stress in HUVEC cells.

a. Cells were cultured at 43°C for 2h, then incubated at 37°C for the indicated times (0h, 1h, 3h, 6h or 9h). Expression levels of phospho-Bcl-2, phosho-ERK1/2 (p-ERK1/2), phospho-JNK1 (p-JNK1) and phospho-p38 kinase (p-p38) were analyzed by Western blotting. Blots were re-probed with total Bcl-2, ERK1/2, JNK1, p38 and β-actin antibody to confirm equal loading of the samples. b. Cells were incubated in the presence or absence of PD98059 (ERK1/2 inhibitor, 25μM), SB203580 (p38 inhibitor, 10μM), or SP600125 (JNK inhibitor, 10 μM), then cultured at 37°C or 43°C for 2h, further incubated at 37°C for 6h. P-Bcl-2 and p-ERK1/2 levels were analyzed by Western blotting. Blots were re-probed with totalERK1/2 and β-actin antibodies to confirm equal loading of the samples. c. Cells were incubated in the presence or absence of PD98059 (25μM), SB203580 10μM), and SP600125 (10 μM), then cultured at 43°C for 2h, further incubated at 37°C for 6h. Cell lysates were immunoprecipitated with an anti-Bcl-2 antibody and immune complexes were analyzed for antibodies by Western blotting. d. Cells were incubated in the presence or absence of MnTBAP (100μM) or catalase (1000 U/μl) for 0.5h prior to heat stress at 43°C for 2hrs, and further incubated at 37°C for 6hrs. LY83583 (10μM) was used as positive control. P-Bcl-2 and p-ERK1/2 were analyzed by Western blotting. Blots were re-probed with total ERK1/2 and β-actin antibodies to confirm equal loading of the samples. Each value represents the mean ± SD of three separate experiments, *P < 0.05, relative to the control group (37°C ), ^# P < 0.05, as compared to the heat stress group (43°C).