Par 4

Antibodies against the following proteins were purchased from the indicated companies: α-Tubulin (Sigma-Aldrich); BAG3 (OriGene); BiP, Caspase-3 (8G10), c-Fos, CHOP, EGR-3, eIF2α, eIF2αx-pS51, FLIP (D16A8), FosB, HSF-1, HSP40, HSP70, IRE1α, PARP, PAR-4, PERK, TAZ, and YAP (Cell Signaling); EGR-1 and Withaferin A (Santa Cruz Biotechnology); GAPDH (Fitzgerald); HSPA6 (Abcam); LC3 (MBL); and vimentin (ARP).

Control and treated cells were lysed using RIPA lysis buffer (120 mM NaCl, 1.0% Triton X-100, 20 mM Tris–HCl, pH 7.5, 100% glycerol, 2 mM EDTA and protease inhibitor cocktail, Roche, Germany). Total protein (30 μg) was electrophoresed on 10% SDS–polyacrylamide gels and blotted onto PVDF membrane (Millipore, Bedford, MA, USA). After blocking with 5% BSA at room temperature, the blots were probed with one of these antibodies: Par-4 (Cell signaling technologies, Santa Cruz Biotechnology, USA), PARP (Santa Cruz Biotechnology), GRP78 (Abcam, Cambridge, England), Akt, PKCζ, actin (Molecular probes, Invitrogen, USA), pAkt-ser 473, or pPKC ζ (Santa Cruz Biotechnology) overnight at 4 °C. The secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG and goat-anti-mouse IgG (Bio-Rad, USA). The immunoreactive bands were visualized by chemiluminescence using Super Signal West Femto Maximum Sensitivity Substrate (Pierce, USA). GAPDH and actin were used to normalize levels of proteins detected.
For secretion of Par-4, HNGC-2, LN-18 and G1 cells were exposed to tamoxifen for 24 h and supernatants were collected and concentration (20-fold) was achieved using 30 kDa cut-off filters (Millipore, USA).