Western Blotting Analysis of Apoptosis-Related Proteins

Western blotting was performed by standard protocols as described previously (Chen et al., 2013b (link); Sun et al., 2014 (link)). Proteins were probed with the following antibodies: SIAH1 rabbit pAb (Abcam, Cambridge, MA, United States), β-Actin mouse mAb (Santa Cruz, Santa Cruz, CA, United States), Phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb (Cell Signaling Technology, Inc., Beverly, MA, United States), p38 MAPK rabbit pAb (Cell Signaling Technology, Inc., Beverly, MA, United States), Phospho-p53 (Ser15) rabbit pAb (Cell Signaling Technology, Inc., Beverly, MA, United States), p53 mouse mAb (Abcam, Cambridge, MA, United States), p53 upregulated modulator of apoptosis (PUMA) rabbit pAb (Abcam, Cambridge, MA, United States), Bcl-2 homologous antagonist/killer (Bak) rabbit pAb (Cell Signaling Technology, Inc., Beverly, MA, United States), and cleaved caspase-3 (Asp175) rabbit pAb (Cell Signaling Technology, Inc., Beverly, MA, United States). The membranes were developed on Molecular Imager ChemiDoc XRS + (Bio-Rad, Hercules, CA, United States), and the intensity of the protein band was analyzed by ImageJ software (1.48V, National Institutes of Health, United States). All Western blot analyses were performed in triplicate.