Enhanced chemiluminescence plus reagent

Cell lysates were analyzed by western blotting as described previously [22] (link). Cell lysate protein was transferred onto Immobilon-P membranes (Millipore; Bedford MA, USA). Blots were blocked in 5% non-fat milk or 5% bovine albumin and then incubated with primary antibody for pS6RP (Cell Signaling), S6RP (Cell Signaling), p4E-BP1 (Cell Signaling), total 4E-BP1 (Cell Signaling), pAkt (Cell Signaling), total Akt (Cell Signaling), cleaved PARP (Cell Signaling), caspase 3 (Cell Signaling), LC3 (Cell Signaling), MCT-1 (Millipore) or MCT-4 (Santa Cruz Biotechnology). The membranes were then incubated with anti-rabbit secondary antibody (GE Healthcare). Western blots for α-tubulin (Cell Signaling) provided a loading control. Specific binding antibody-target protein interactions were detected using enhanced chemiluminescence plus reagents (Amersham Biosciences, Buckingham-shire, UK) and exposure to either Hyperfilm ECL (Amersham) or XOMAT Kodak (Rochester NY, USA) autoradiography film.

The expression of target proteins, including cochlin, p-CaMKII, CaMKII, p-β-catenin, and β-catenin, was examined by western blots (n = 4–16/group). Briefly, 50 μg of total protein from each sample and a multicolor prestained protein ladder (Cat# WJ103, EpiZyme, Shanghai, China) were resolved in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The blots were washed, blocked with 5% nonfat dry milk or BSA, and probed with the corresponding primary antibodies (Supplementary Table 3) at 4 °C overnight. On the next day, the blots were washed and incubated with an appropriate secondary antibody (Supplementary Table 3) at RT for 2 h. The protein signals were visualized with enhanced chemiluminescence plus reagents (Amersham Biosciences, Piscataway, NJ, USA) and imaged using a Multispectral Imaging System (UVP, LLC, Upland, CA, USA). The optical densities of cochlin, p-CaMKII, CaMKII, p-β-catenin, and β-catenin were quantified using ImageJ (National Institute of Health, Bethesda, MD, USA) and normalized to that of an internal standard, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The ratios of p-CaMKII/CaMKII and p-β-catenin/β-catenin were also calculated.

Cells were harvested and suspended in RIPA lysis buffer (Thermo, Hercules, CA) containing a protease inhibitor cocktail (Sigma–Aldrich Corporation, St Louis, MO). After incubation on ice for 15 min, cell lysates were centrifuged at 13 000 r.p.m. for 15 min at 4°C. The protein content of the supernatant was determined using the Thermo Protein Assay Reagent (Thermo, Hercules, CA). 30-60 μg Proteinsper well were separated by 12% sodiumdodecyl sulfate–polyacrylamide gel electrophoresis and transferred topolyvinylidene difluoride membranes (Bio-Rad Laboratories). The following primary antibodies were used to probe the alterations of protein:LMP1(CS1-4, Dako), PD-L1(E1L3N™, Cell Signaling Technology, Danvers, MA), p-stat3, total-stat3, p-NF-κB (P65), NF-κB (P65), p-c-fos, p-c-Jun, p-JAK3, JAK3, p-ERK1/2, ERK1/2 (Cell Signaling Technology, Danvers, MA) and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were detected by enhanced chemiluminescence Plus reagents (Amersham Pharmacia, Piscataway, NJ). Signal quantification was obtained using Quantity One software (Bio-Rad Laboratories, Hercules, CA) and normalized to β-actin.

24 h after transfection, cells were incubated with serum-free medium for 16 h. Cells were then stimulated with 10 μg/ml collagen I for 90 min at 37 °C and lysed in 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris, pH 7.4, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 1 mm sodium orthovanadate, 5 mm NaF, and 10 mm NEM. Aliquots of the lysates were analyzed by nonreducing SDS-PAGE on 5% polyacrylamide gels followed by blotting onto nitrocellulose membranes. The blots were probed with anti-DDR1 Abs followed by horseradish peroxidase-conjugated goat anti-rabbit secondary Abs. Signal detection was performed using Enhanced Chemiluminescence Plus reagent (Amersham Biosciences) on an Ettan DIGE Imager (GE Healthcare). Intensities of protein bands were quantitated using ImageQuant TL (GE Healthcare).

The samples (60 μg protein/lane) were electrophoresed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then electrophoretically transferred onto Hybond-C Extra membranes (Amersham, Pittsburgh, PA, USA). Following treatment with the blocking buffer, the membranes were incubated with primary antibodies (anti-t-Akt and anti-p-Akt; anti-t-eNOS and anti-p-eNOS) and the horseradish peroxidase-conjugated secondary antibody (Sigma). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The immunoreactive bands were detected by Enhanced Chemiluminescence Plus reagent (Amersham) using an X-ray film (Kodak; Rochester, NY, USA). Target signals were normalized relative to the GAPDH expression and assessed using ImageJ 1.36 software.