Western Blot Analysis of CIDEA Regulation

Western blot analysis was performed with whole lysates, cytosolic and nuclear protein extracts of cells transfected with CIDEA overexpressing plasmid or treated with inhibitors of PPARγ, HIF-1α or JNK as described,^{33 (link)} using antibodies against CIDEA (Abcam, Cambridge, UK), PPARγ, HIF-1α (Novus Biological, Cambridge, UK), phospho-JNK, JNK, phospho-cofilin, Cofilin, phospho-VASP, VASP, phospho-STAT3 (Y705), STAT3, p53, acetyl-p53 (Lys-373 and Lys-382) (Millipore, Billerica, MA, USA); Cyclin B1, β-tubulin, GAPDH, C-23 and NFκB Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies were purchased from Cell Signaling (Danvers, MA, USA) unless otherwise mentioned. After addition of horse radish peroxidase-conjugated secondary antibodies (Vector Laboratories Inc., Burlingame, CA, USA) blots were exposed to Chemigenius Bioimaging System (Syngene, Cambridge, UK) and images were developed by Gene snap software (Syngene). Reprobing of the blots was performed after stripping to determine the loading control with anti-β-tubulin, GAPDH or C23 antibodies.