Ab178547

Proteins were extracted from the frozen lung tissues by grinding with protease inhibitors. The proteins were incubated on ice for 30 min. The protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were incubated with 5% non-fat milk for 1 h, and then incubated with primary antibodies. WISP1 of primary rabbit monoclonal antibody (ab178547, Abcam, USA) was used for Western blot analysis. Membranes were detected using the Odyssey Two-Color Infrared Laser Imaging System (LI-COR Biosciences).

Total protein content in tissues or cells was extracted by radio-immunoprecipitation assay lysis buffer containing phenylmethylsulfonyl fluoride. Protein concentration was determined by a bicinchoninic acid kit. Next, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Membranes were then blocked with 5% skim milk powder for 1 h at room temperature and incubated with the primary antibodies (Abcam Inc., Cambridge, MA, USA) of rabbit anti-mouse antibodies to WISP1 (ab178547, dilution ratio of 0.5 μg/mL), caspase-1 (ab1872, dilution ratio of 1: 1000) and GAPDH (ab9485, dilution ratio of 1: 2500, internal reference) overnight at 4 °C. Membranes were then washed with Tris-buffered saline Tween-20 and further incubated with the horseradish peroxidase-conjugated secondary antibody of goat anti-rabbit IgG (ab97051, dilution ratio of 1: 2000; Abcam Inc., Shanghai, China) for 1 h at room temperature. Proteins on the membrane were visualized by enhanced chemiluminescence detection kits (BB-3501, Amersham Pharmacia Biotech, UK) and Bio-Rad image analysis system (Bio-Rad Laboratories, Inc. CA, USA). The protein band intensity was determined using the Quantity One v4.6.2 software. The ratio of gray value of target protein band to that of GAPDH was regarded as the relative protein expression.