Amersham ecl western blotting detection reagent

For the analysis of protein expression, cell pellets were washed and resuspended in PBS. Cells were centrifuged and pellets were stored at −20 °C. Cell pellets were then lysed in Winman’s buffer containing 1% NP-40, 0.1 M Tris–HCl pH 8.0, 0.15 M NaCl, and 5 mM EDTA, complemented with protease inhibitor cocktail (Roche). The total protein content was quantified using the DC™ Protein Assay kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions. Protein lysate (20 µg) were loaded on 12% SDS-PAGE gel and transferred into a nitrocellulose membrane (GE Healthcare, Cleveland, OH, USA). The following primary antibodies were used: rabbit anti-PARP-1 (1:2000, sc-7150, Santa Cruz Biotechnology, Heidelberg, Germany), mouse anti-Caspase 3 (1:2000, 05-654, Merck Millipore, Darmstadt, Germany), and goat anti-actin (1:2000, sc-1616, Santa Cruz Biotechnology). The corresponding secondary antibodies were: anti-rabbit IgG-HRP, anti-mouse IgG-HRP, or anti-goat IgG-HRP (1:2000, Santa Cruz Biotechnology). The Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the High Performance Chemiluminescence Film (GE Healthcare), and the Kodak GBX developer and fixer (Sigma) were used for signal detection [35 (link),36 (link)].

Western blot analyses were performed as previously described [37 (link)]. Briefly, the cell lysates were prepared in a modified RIPA buffer: 20 mM Tris, pH 7.4, 100 mM NaCl, 0.1% SDS, 1% NP-40, 1 mM EDTA, 1% Triton X-100, 0.5% deoxycholate, phosphatase inhibitor cocktail (phosSTOP, Roche, Basel, Switzerland; 04-906-837-001), and protease inhibitor cocktail (cOmplete tablets, Roche, 04-693-116-001). The cell lysates were centrifuged at 13,000 rpm for 30 min. The protein concentration in the supernatant was determined using a Bio-Rad protein assay reagent (Bio-Rad, Richmond, CA, USA) according to the manufacturer’s instructions. Equal amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). The blots were blocked overnight with 10% skim milk in TPBS at 4 °C and probed overnight with the primary antibodies listed above. After secondary antibody incubation, specific bands were detected using Amersham ECL western blotting detection reagents (GE Healthcare, Piscataway, NJ, USA).

CFs were washed with PBS (0.1 mol/L, pH 7-7.4) three times, lysed in a lysis buffer for 30 min at 4 °C, and then scraped off using a cell scraper before being mixed with a one-quarter volume of 5× loading buffer. The cell protein mixture was then boiled in a 100 °C water bath and cooled to room temperature. The protein concentration was determined using a Bradford protein assay kit (Amresco, M173-KIT). Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The strips were blocked with 5% nonfat milk for 1 h at room temperature. The strips were then incubated overnight with primary antibodies against AAT1 (Sigma, AV48205, 1:500), collagen I (Abcam, ab254113, 1:500), collagen III (Abcam, ab7778, 1:500), α-SMA (Abcam, ab5694, 1:500), β-catenin (Abcam, ab6302, 1:500), p-P38 MAPK (Abcam, ab4822, 1:500), P38 MAPK (Abcam, ab170099, 1:500) and GAPDH (Abcam, ab181602, 1:2000) diluted in PBS with 0.05% Tween 20 at 4 °C. The strips were rinsed using PBS with 0.05% Tween 20 for 30 min, followed by incubation with secondary antibodies for 1 h at room temperature. All protein bands were detected using Amersham ECL Western blotting detection reagents (GE Healthcare, RPN2106) and analyzed with a biological electrophoresis image analysis system.

Cells were seeded in 6 cm dishes and allowed to adhere overnight. Following the relevant experiments, cells were lysed on ice in RIPA buffer (1 mM EDTA; 1% v/v NP40; 0.5% w/v sodium deoxycholate; 0.1% v/v SDS; 50 mM sodium fluoride; 1 mM sodium pyrophosphate in PBS, SigmaAldrich) containing phosphatase (PhosphoSTOP, Roche) and protease (Complete ULTRA, Roche) inhibitors. Protein concentrations were quantified using the DC Protein assay (Bio-Rad). Equivalent amounts of protein lysates were boiled, resolved by SDS-PAGE using 10% w/v acrylamide gels, and transferred to polyvinylidene difluoride membranes. Membranes were incubated for 1 h in blocking buffer (5% w/v skim milk, 0.05% v/v Tween-20 in Tris-buffered saline; TBS-T) and probed overnight at 4 °C with the primary antibody. Blots were washed thrice in wash buffer (0.05% v/v TBS-T) for 10 min, followed by incubation with peroxidase-conjugated secondary antibody (Dako) for 1 h at room temperature. Proteins were visualised by Amersham ECL western Blotting Detection Reagents (GE Life Sciences) or ECL Plus western blotting substrate kit (Thermo Scientific). Anti-β-actin or anti-GAPDH antibodies were used to assess protein loading. Antibodies are detailed in Supplementary Table 3.

Bradford DC protein assay (Bio-Rad, USA) was used to measure the concentration of protein lysates. Subsequently, 20–40 μg of protein was resolved on a 12% Bis-Tris polyacrylamide gel, electrotransferred onto nitrocellulose membranes (GE Healthcare, Piscataway, USA) and blocked with 5% nonfat-milk. Protein blots were immunostained overnight with primary antibodies at 4°C and for 1 hour with secondary antibodies at room temperature. Amersham ECL Western Blotting Detection Reagents (GE Healthcare) were used to visualize the protein signal.