Ecl plus chemiluminescent reagent

The liver tissue were dissolved in ice-cold lysis buffer (1:10), and homogenized using QIAGEN Tissuelyser LT (5 min, 50 Hz) followed by centrifugation at 13,000 rpm for 10 min at 4°C. Protein concentration was determined using the BCA assay (Thermo Scientific, Rockford, IL, USA). The proteins were resolved in 8% SDS-PAGE and transferred to PVDF membranes. The PVDF membrane was blocked in a 5% BSA-TBST buffer for 1 h at room temperature, then incubated with the primary antibody (1:1,000) at 4°C overnight. After washing three times, the membrane was incubated with the secondary antibody for 1 h at room temperature. The protein signals were detected in the membrane using the ECL Plus chemiluminescent reagent (GE Heathcare UK Ltd., Little Chalfont, Buckinghamshire, UK). The protein signals of interest were collected using the Bio-Rad ChemiDoc™ XRS imaging system.

Protein was isolated from homogenized lung tissue using a lysis buffer consisting of 50 mM Tris·HCl (pH 7.4–7.6), 150 mM NaCl, 1% Igepal CA-630, 10 mM NaF, 0.5% sodium deoxycholate, 2 mM sodium orthovanadate (all Sigma), and a protease inhibitor cocktail (Roche). Lysed protein was quantified using the Lowry method (Bio-Rad), run on SDS-PAGE, and transferred to Hybond PVDF membrane (GE Healthcare). Immunoblotting was performed using primary antibodies to mouse angiopoietin-2 (clone: F-18, Santa Cruz, Dallas, TX) and α-tubulin (clone: DM1A, Sigma-Aldrich, St. Louis, MO), horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (both Dako), and visualized using ECL Plus chemiluminescent reagent (GE Healthcare). The specificity of the angiopoietin-2 antibody was validated previously by Abbott and Buckalew (1 (link)).