Enhanced chemiluminescence detection kit

Total proteins were extracted from Tan I-treated H28 cells using lysis buffer (Institute de Biologie Structurale-BR002) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and protease inhibitor cocktail). The proteins were separated on 10–12.5% SDS-PAGE gels and transferred to Hybond ECL transfer nitrocellulose membranes (GE Healthcare Bio-Science). After blocking with 5% nonfat dry milk, the membranes were incubated with the desired primary antibodies [p62 (5114S), LC3II (3868S), poly(ADP)-ribose polymerase (PARP) (12061), CHOP (2898S), PERK (12185), ATF4 (11815S), ATG5 (FL-25), Beclin-1 (4445), IRE1 (3294), β-Actin (12262) (Cell signaling), and ATF6 (Santa Cruz Biotechnology, sc22799)], followed by a horseradish peroxidase-labeled anti-rabbit (sc2313) or mouse IgG (sc10198). The immune-reactive bands were visualized with an enhanced chemiluminescence detection kit (Amersham Pharmacia).

After the treatment, the cells or the medium were obtained. For cellular protein, the cells were lysed in 0.1 ml of lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 mM DTT, 50 mM sodium fluoride and 1 mM sodium orthovanadate) for 30 min at 4°C. After centrifugation, the supernatants were collected and the concentration of protein was quantified. For protein in the medium was precipitated by 20% TCA for 30 min on ice. After centrifugation for 20 min at 12,000 rpm, the pellets were washed twice by acetone. For Western blot analysis, 30 μg proteins were separated by electrophoresis in a 10% or 14% polyacrylamide gel and transferred to a PVDF membrane. After 1 h incubation at room temperature in PBS/5% non-fat milk, the membrane was washed with PBS/0.1% Tween 20 for another 1 h and overnight incubated with the indicated antibody at 4°C. After three washings with PBS/0.1% Tween 20, the anti-mouse or anti-rabbit IgG (dilute 1:8000) was applied to the membranes for 1 h at room temperature. The membranes were washed with PBS/0.1% Tween 20 for 1 h. The detection of signal was performed with an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, United Kingdom) and the membranes were scanned using a ChemiDoc^TM MP Imaging System.

Cells were lysed in ice-cold buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1% triton X-100) containing protease/phosphotase inhibitor cocktails (Sigma-Aldrich). Protein concentration was quantified using the BCA protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with 5% fat-free milk at room temperature for 1 h to block nonspecific binding, and probed with the primary antibodies recognizing cyclin D1, FOXO3, and GAPDH (Cell Signaling Technology, Inc., Beverly, MA, USA). After washing, the blots were incubated with peroxidase-labeled secondary antibodies (Cell Signaling Technology) and developed using an enhanced chemiluminescence detection kit (Amersham Biosciences, Inc., Piscataway, NJ, USA). The intensities of the bands were quantified by densitometry using Quantity One software (Bio-Rad Laboratories).

Extraction of proteins from mouse colonic mucosa and Western blot analysis were performed as previously described [40 (link),41 (link)]. The primary antibodies used were anti-LC3 (#L8918, Sigma-Aldrich, Saint-Louis, MO, USA), anti-phospho-H2AX (#2577, Cell Signaling) and anti-α-tubulin (#2144, Cell Signaling). The secondary antibody used was HRP-conjugated anti-rabbit (#7074, Cell Signaling). Blots were detected using the Enhanced Chemiluminescence Detection kit (RPN2108, Amersham Biosciences, Buckinghamshire, UK) and revealed using the ChemiDocTM XRS System (BioRad, Hercules, CA, USA).

Western blot analysis was carried out for VEGFR2 using an anti-VEGFR2 antibody (Abcam Co.) as the primary antibody. The cells were collected and lysed by RIPA lysis buffer (Sigma-Aldrich Corp., St. Louis, MO, USA). Total protein was extracted and stored at -80˚C. The extracts were then mixed with 6× sodium dodecyl sulfate (SDS) buffer and boiled for 4 minutes. Samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% (w/v) skim milk in PBS that contained 0.1% Tween-20 for 1 hour at room temperature, washed with PBS, and probed with primary antibodies overnight at 4˚C. Membranes were washed again with PBS and incubated at room temperature with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies (Abcam Co., UK) for 1 hour. Proteins were visualized with an enhanced chemiluminescence detection kit (Amersham Bioscience, Buckinghamshire, England). Actin (a goat polyclonal antibody) was used as the internal control.