Horseradish peroxidase conjugated anti rabbit or mouse igg secondary antibody

Cells or lung tissues were lysed in a lysis buffer (Cell Signaling), briefly sonicated, incubated on ice for 30 min, and then centrifuged at 12,000×g for 10 min. Supernatants were collected and stored at – 30 °C until use. For protein fractionation, 10 µg of each cell lysate was loaded on gels and proteins were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Life Technologies) and transferred onto nitrocellulose membranes. After overnight incubation with a primary antibody, the membranes were washed and incubated with horseradish peroxidase-conjugated anti-rabbit or -mouse IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The presence of proteins of interest was visualized and quantitated with C-DiGit Blot scanner (Scrum, Tokyo, Japan). Blots were photographed, digitized and the density of each band was analyzed using Image J, a public domain software developed by the NIH.

Cells or lung samples were lysed in lysis buffer (Cell Signaling), briefly sonicated, incubated on ice for 30 minutes, and centrifuged at 12,000 g for 10 minutes. Supernatants were collected and stored at –80°C until use. For protein fractionation, 10 µg cell lysate was loaded per lane on gels for fractionation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Life Technologies), and transferred onto a nitrocellulose membrane. After overnight incubation with a primary antibody, the membrane was counter-stained with horseradish peroxidase-conjugated anti-rabbit or -mouse IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and visualized with an enhanced chemiluminescence system (Cell Signaling). Blots were photographed, digitized and analyzed using Image J, a public domain software developed by the NIH.