Anti bad

Anti-GAPDH, anti-phospho-JNK1/2, anti-β-actin, anti-caspase-3, anti-caspase-7, anti-caspase-9, anti-poly(ADP-ribose) polymerase (PARP), anti-Bax, anti-Bad, anti-Bcl-2, anti-cytochrome c, anti-Akt, and anti-COX IV antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). SP600125, Z-VAD-FMK, MitoTEMPO, surfactin, and urban PM (SRM 1648a) were purchased from Sigma (St. Louis, MO, USA). N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI) were taken from Biomol (Plymouth Meeting, PA).

Total protein was extracted from cell samples by using RIPA buffer (Beyotime). 50 μg of the extracted and the co-precipitated proteins were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk for 2 h at room temperature, the membranes were incubated with anti-p-Bad (1:200, Santa Cruz), anti-p-AKT (1:200, Santa Cruz), anti-PTEN (1:200, Santa Cruz), anti-Bad (1:200, Santa Cruz), anti-AKT (1:200, Santa Cruz), anti-Bcl-2 (1:200, Santa Cruz), anti-cytochrome c (1:200, Santa Cruz), anti-caspase-9 (1:200, Santa Cruz) and anti-caspase-3 (1:200, Santa Cruz). After incubation with primary antibodies, membranes were washed and incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz) for 2 h at room temperature. Proteins on the membrane were visualized by using an enhanced chemiluminescence detection kit (Pierce, Rockford, IL, USA). Glyceraldehyde-3-phosphate (GAPDH) was used as an internal control to ensure equal protein loading. Mitochondria/Cytosol Fraction Kit (BioVision, Milpitas, CA, USA) was used to separate the mitochondria fraction and cytosol fraction before detection of cytochrome c.