caveolin-1 Antibody (7C8): sc-53564

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:400) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:400) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

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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Manufacturer protocol

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