C ebp homologous protein chop

Cells were lysed in radio-immunoprecipitation assay buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% Triton X-100) at 4 °C. Protein concentrations in the lysates were measured using a DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). Proteins in aliquots of the lysate were separated by 12% SDS-PAGE and electro-transferred to PVDF membranes (Immobilon-P; Millipore, Bedford, MA, USA), using a Bio-Rad Semi-Dry Transfer Cell. The blots were then incubated with primary antibodies against α-actinin (ACTN), p53, glucose-regulated protein 78 (GRP78) (Santa Cruz Biotechnology, Dallas, TX, USA), C/EBP homologous protein (CHOP) (Cell Signaling, Danvers, MA, USA), cyclin D1, and p21 (Abcam, Cambridge, UK). Thereafter, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The immunoreactive proteins were detected using ECL^TM Western Blotting Detection Reagent and Amersham Hyperfilm^TM ECL (GE Healthcare, Waukesha, WA, USA). The procedural details were described in our previous publications [33 (link),34 (link)].

After homogenization of the cells, the protein concentration was measured using the Bradford protein assay (Bio-Rad, CA, USA). Protein (25 μg) was loaded into the wells, separated on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels (10–15%) (80 V, 3 h), and transferred to a nitrocellulose membrane (50 V, 2 h). After membranes were blocked in 5% skim milk for 1 h, immunoblotting was conducted by incubating with the primary antibodies overnight at 4°C. In this study, 78-kDa glucose-regulated protein (Grp78), binding immunoglobulin protein (Bip), inositol-requiring protein 1 (IRE-1), phospho-IRE-1α, C/EBP homologous protein (CHOP), and microtubule-associated protein-1 light-chain 3 (LC3) antibodies were purchased from Cell Signaling (MA, USA). Sequestosome 1 (SQSTM1, p62), voltage-dependent anion-selective channel protein 1 (VDAC1), and beclin-1 (BECN1) antibodies were purchased from Abcam (MA, USA). Actin and 75 kDa glucose regulated protein (Grp75) antibodies were purchased from Santa Cruz (CA, USA). After incubation with HRP-conjugated secondary antibodies for 2 h at room temperature and washing, the bands of interest were measured using an ATTO CS image analyzer 3.0 (ATTO Corp., Tokyo, Japan) [17 (link)].

Cantharidin (chemically pure compound), 3-(4,5-dimethyl thiazol-2-yl-)-2,5-diphenyl tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies used in the this study were directed against the following: caspase-9, caspase-7, caspase-3, glucose-regulated protein (Grp)78, eIF-2α, phospho-eIF-2α, cytochrome c, AIF, Bax, Bid, Bak, Bcl-2, JNK, phospho-JNK, p38, phospho-p38, extracellular signal-regulated kinase (ERK), phospho-ERK (Santa Cruz Biotechnology, USA), caspase-12 (Becton Dickinson, San Jose, CA, USA), Grp94, and C/EBP homologous protein (CHOP) (Cell Signaling Technology, USA). The cell culture medium included Dulbecco's modified Eagle's medium with 45% Ham's F12 medium and 10% fetal calf serum, all purchased from Gibco/Invitrogen (Carlsbad, CA, USA). The chemiluminescence reagents were purchased from Amersham Biosciences, Sweden. All other chemical reagents which were no specified in this study were obtained from Sigma-Aldrich.

Lysates were prepared from tumour cells and analysed by western blotting. Primary antibodies include cleaved caspase-3 (1:2000, Cell Signalling #9661), phospho-rpS6 (Ser240/244) (1:5000, Cell Signalling #5364), rpS6 (1:5000, Cell Signalling #2217), phospho-p70 S6K (Thr389) (1:1000, Cell Signalling #9205), p70 S6K (1:1000, Santa Cruz #sc-230), C/EBP homologous protein (Chop) (1:1000 Cell Signalling #2895), FIP200 (1:1000, Cell Signalling #12436), p62 (1:2000, Cell Signalling #39749), LC3B (1:2000 Cell Signalling #3868), Raptor (1:1000, Cell Signalling #2280), Rictor (1:1000, Cell Signalling #2114), acetyl-histone H3(Lys9) (1:2000, Cell Signalling #9649), vinculin (1:10,000, Sigma-Aldrich #V4505) and NRF2 (1:1000, GeneTex #103322). Horse radish peroxidase (HRP)-linked anti-rabbit immunoglobulin G (IgG) or anti-Mouse IgG (1:10,000, Cell Signalling) were used as secondary antibodies. WesternBright ECL HRP substrate (Advansta) was used for signal imaging.

CWP232291 was obtained from JW Pharmaceutical Corporation (Seoul, Korea). The compound z-valine-alanine-aspartate-fluoromethylketone (ZVAD-FMK) was purchased from R&D Systems (Minneapolis, MN). The following primary antibodies were used: cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), phospho-eIF2a serine 51 [peIF2a (serine-51)], eIF2a, inositol-requiring kinase 1 (IRE1), C/EBP-homologous protein (CHOP) (Cell Signaling Technology, Danvers, MA), AR, survivin, GAPDH, β-actin, bcl-2 (Santa Cruz Biotechnology, Dallas, TX), β-catenin (Merck Millipore, Burlington, MA) and AR-V7 (Precision Antibody, Columbia, MD).