Publication protocol
Following collection of BALF, the lung tissues were washed in ice-cold saline, then homogenized in 4°C RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) with 1 mM phenylmethanesulfonyl fluoride and centrifuged at 3,000 × g and 4°C for 15 min. The supernatants were collected and the protein concentration was determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). Protein samples (40 µg) were loaded per lane and separated using 10% SDS-PAGE (Beyotime Institute of Biotechnology). The target proteins, including phosphorylated Src, VE-cadherin and caveolin-1, were then electrophoretically transferred onto nitrocellulose membranes (Beyotime Institute of Biotechnology). The protein blots were blocked in Tris-Buffered Saline and Tween 20 (TBST; 5% non-fat milk, 10 mM Tris, 150 mM NaCl, 0.05% Tweek-20) for 1 h, followed by incubation with primary antibodies against phosphorylated Src (monoclonal; 1:200, rabbit anti-mouse; ab4816; Oncogene Research Products; La Jolla, CA, USA), phosphorylated VE-cadherin (polyclonal, 1:400; rabbit anti-mouse; SAB4504676; BD Biosciences, Franklin Lakes, NJ, USA), VE-cadherin (monoclonal; 1:400; rabbit anti-mouse; V1514; BD Biosciences), phosphorylated caveolin-1 (polyclonal, 1:400; rabbit anti-mouse; sc-14037; Santa Cruz Biotechnology Inc., Dallas, TX, USA) or caveolin-1 (monoclonal; 1:400; rabbit anti-mouse; sc-53564; Santa Cruz Biotechnology Inc.) overnight at 4°C. Blots were then treated with the following secondary antibodies in TBST solution for 1 h: Secondary Src antibody [polyclonal; 1:4000; chicken anti-rabbit immunoglobulin G (IgG); ab6829; Abcam, Cambridge, MA, USA], phosphorylated VE-cadherin (polyclonal), VE-cadherin (monoclonal), phosphorylated caveolin-1 (polyclonal) or caveolin-1 (monoclonal; all 1:3,000; chicken anti-rat; ab112448; Abcam). Each sample was also probed with β-actin antibody (1:30,000; rabbit anti-mouse; A5316l; Sigma-Aldrich, St. Louis, MO, USA) as a loading control, and β-actin secondary antibody (monoclonal; 1:3,000; chicken anti-mouse IgG; ab131368, Abcam). Finally, blots were washed with PBS with Tween 20 and then examined using the ECL Plus Western Blotting Detection System (Amersham Life Science, Little Chalfont, Buckinghamshire, UK).
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