Anti creb 2

Cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8. Protein content of cell lysates was measured using Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA, USA, 23225). During each procedure equal amounts of protein were used. SDS-PAGE was done by using Hoefer miniVE (Amersham, UK). Proteins were transferred onto Millipore (Billerica, MA, USA) 0.45 µm PVDF membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and for antibody solutions. Loading was controlled by developing membranes for GAPDH or dyed with Ponceau S in each experiment. The following antibodies were applied: antiLC3B (SantaCruz, Santa Cruz, CA, USA, sc-16755), antiPARP (Cell Signaling, Danvers, MA, USA 9542S), antiGADD 153 (SantaCruz, sc-7351), antiCREB-2 (SantaCruz, sc-200), antiP-c-Jun (Cell Signaling, 9261S), antic-Jun (Cell Signaling, 9165S), antiPERK (Cell Signaling, 3192S), antiULK1 (Cell Signaling, 8054S), antiP-ULK1 (S555) (Cell Signaling, 5869S), antieIF2α (Cell Signaling, 9722S9), antiP-eIF2α (Cell Signaling, 9721L), and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).

Cellular extracts (20 μg) were migrated in a 12% SDS-PAGE and transferred on a PVDF membrane (Millipore). Membranes were blocked with 5% powder milk and incubated with anti-JunD (1 μg/ml), anti-CREB2 (1 μg/ml), anti-Syncytin-2 (1/7500) or anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1/500) antibodies (Santa Cruz Biotechnology Inc., Dallas, TX). After several washes, membranes were further incubated with appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibodies (1/10,000) (Cell Signalling, Danvers, MA), and signals were detected using the BM chemiluminescence blotting substrate (Roche Diagnostics). Membranes were scanned with the Fusion FX5 system (Montreal Biotech Inc., Dorval, Canada) and band intensities were determined and normalized with GAPDH with the Quantity One 4.5.0 image acquisition software.

Lysates of CL tissue from each stage were prepared in PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea). The protein concentration for each sample was estimated using a Bradford dye-binding assay with bovine serum albumin (BSA) as a standard (Bradford, 1976). Aliquots of the proteins (30 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels. After electrophoresis, the separated proteins were transferred onto nitrocellulose (NC) membranes (Pall Corporation, Port Washington, NY, USA). After blocking with 5% non-fat milk in Tris-buffered saline (TBS) with 0.1% Tween 20 at 4 °C under mild agitation, the membranes were incubated with 1:2000 diluted anti-CHOP, anti-p90ATF6, anti-CREB-2, anti-3β-HSD (Santa Cruz, Biotechnology, CA, USA), anti-GRP78/BIP, anti-p-eIF2α, anti-IRE1, anti-eIF2a, anti-JNK, anti-p-SAPK/JNK, anti-caspase-3, anti- β-tubulin (Cell Signaling, Beverly, MA, USA), anti-p50ATF6 (IMG, San Diego, CA, USA), and anti-p-IRE1α (Abcam, Cambridge, MA, USA) antibodies. The membranes were then incubated with secondary horse radish peroxidase (HRP)-conjugated anti-goat/mouse/rabbit IgG (Thermo, Rockford, IL). Antibody binding was detected using an enhanced chemiluminescence (ECL) kit (Advansta, CA, USA). Band intensities were quantified using the Image J software (NIH, MD, USA).