β-catenin Antibody (E-5): sc-7963

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -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 (1:500) -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

Protein lysates were extracted from SMCs or blood vessels using RIPA buffer (50 mM
Tris-HCl pH 7.4, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 150
mM NaCl) plus protease inhibitors (Complete mini Roche Life Science 04693159001). A
BCA protein assay kit (Pierce 23335) was used to measure protein concentrations, and
equal amounts of protein were loaded (20 – 60 µg) and separated by 10%
polyacrylamide gel electrophoresis. Proteins were transferred (Trans-Blot SD cell, BIORAD 170-3940) to 0.45 µm pore size PVDF membranes (Immobilon-P, Millipore),
blocked for 1 hour at room temperature with TBST (Tris pH8.0, NaCl 150 mM, 0.1 %
Tween 20) plus 4 % (wt/vol) nonfat milk, and incubated overnight at 4 ºC with anti-βcatenin (Santa Cruz sc-7963, 1:500 dilution), anti-GAPDH (Santa Cruz sc-25778,1:5000 dilution), anti-PDGFRβ (Cell signaling 3165, 1:1000 dilution), anti-Timp1 (Thermo fisher scientific PA5-13668, 1:2000 dilution), anti-Timp2 (Thermo fisher scientific MA1-774, 1:2000 diution), anti-Mmp2 (Thermo fisher scientific PA1-1667,
1:1500 dilution), anti-N-cadherin (Cell signaling 4061, 1:500 dilution), or anti-β-actin (Abcam 8226, 1:5000 dilution) as primary antibody in blocking solution. After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour at
8 room temperature. After washing, signals were detected by adding ECL substrate (ECL
Western Blotting Substrate, Pierce 32106) and exposing films to membranes. Densitometric analysis was performed with Image J.

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Manufacturer protocol

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