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Western blotting and co-immunoprecipitation

CC Cheng-Wei Chou
CL Ching-Heng Lin
TH Tzu-Hung Hsiao
CL Chia-Chien Lo
CH Chih-Ying Hsieh
CH Cheng-Chung Huang
YS Yuh-Pyng Sher
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The following antibodies were used for western blotting: p53 (OP43; Merck Millipore), MDM2 (SMP14) (sc-965; Santa Cruz), AKT (2920; Cell Signaling Technology), phospho-AKT (Thr308) (9275; Cell Signaling Technology), phospho-AKT (Ser473) (9271; Cell Signaling Technology), PARP (9532; Cell Signaling Technology), caspase 3 (9661; Cell Signaling Technology), phospho-5′ AMP-activated protein kinase ([AMPK] bs-4002R; Bioss), AMPK (E-AB-30490; Elabscience Biotechnology Inc.), phospho-mammalian target of rapamycin ([mTOR] (E-AB-20929; Elabscience Biotechnology Inc.), mTOR (E-AB-32129; Elabscience Biotechnology Inc.), LC3A/B (4108; Cell Signaling Technology), α-tubulin (NB100-690; Novus Biologicals), GAPDH (10494-1-AP; Proteintech), HSP-40 (sc-59554; Santa Cruz Biotechnology), CHIP (sc-133066; Santa Cruz Biotechnology), and vinculin (GTX109749; GeneTex). For co-immunoprecipitation assay, cell lysates were incubated with p53 antibody (OP43, EMD Millipore, Billerica, MA) or HSP-40 antibody (sc-398766, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. Then samples were precipitated with appropriate protein A/G beads with matched IgG as negative control. The precipitants were analysed in SDS-PAGE western blot.

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