Hrp conjugated swine anti rabbit secondary antibody

After electrophoresis on 12% acrylamide gel, proteins were transferred on nitrocellulose PVDF membrane (Perkin Elmer Life Sciences, Waltham, MA, USA). Primary antibodies used were rabbit anti-ADAM10 (Abcam; 1/1000) or rabbit anti-N-cadherin (4061, Cell signalling; 1/500). Revelation was performed by chemiluminescence after incubation with HRP-conjugated swine anti-rabbit secondary antibody (Dako, 1/3000). Actin or GAPDH were detected as a loading control.

Lung tissue powder was homogenized in RIPA buffer: 25 mmol/L Tris·HCl,pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, containing Complete® cocktail (Roche). Protein concentration was measured with the bicinchoninic-acid assay (Pierce, Rockford, IL, USA). 25 μg protein was separated on an SDS-polyacrylamide gel, transferred onto 0.45 μm nitrocellulose membranes, using a wet transfer system (Biorad, Hercules, CA, USA), stained with Ponceau S to confirm equal loading of lanes, washed with TBS (50 mmol/L Tris, 150 mmol/L NaCl, pH 7.6) and blocked with 5% skimmed milk in TBS/ 0.5% Tween-2. Arginase 1 was visualized with rabbit anti-arginase 1 antibody (1:200), followed by an HRP-conjugated swine anti-rabbit secondary antibody (DAKO). The signal was developed using the Super Signal West Pico Substrate (Pierce) and quantified with the Fuji systems darkbox (Fuji Film Life Sciences, Tokyo, Japan).

Western blot analysis was performed as described previously [33 (link)]. In brief, tissue powder was homogenized in RIPA buffer (25 mM Tris·HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% Na-deoxycholate, 0.1% SDS, containing Complete® cocktail (Roche). Protein concentration was measured with the bicinchoninic-acid assay (Pierce, Rockford, IL, USA). Twenty-five μg protein was separated by SDS-PAGE, transferred onto 0.45 μm nitrocellulose membranes, using a wet transfer system (Biorad, Hercules, CA, USA). Equal loading of lanes was confirmed by Ponceau S staining, followed by a wash-step with TBS (50 mM Tris, 150 mM NaCl, pH 7.6) and blocking of non-specific binding with 5% skimmed milk in TBS/0.5% Tween-2. Rabbit anti-ARG1 antibody (1:200), followed by an HRP-conjugated swine anti-rabbit secondary antibody (DAKO) was used to visualize ARG1. Signal was developed with the Super Signal West Pico Substrate (Pierce) and quantified in a Fuji systems darkbox (Fuji Film Life Sciences, Tokyo, Japan).