Integrin β1 Antibody (102DF5): sc-73610

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1500) -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 (1:1500) -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

For immunoblotting, we followed standard protocols (58). The 0.5 M NaCl and SDS extracts of the 12 endarterectomy samples were aliquoted and mixed with denaturing sample buffer and boiled at 96°C for 10 minutes. On separate 15-well 4%–12% polyacrylamide gels (NuPAGE, Invitrogen) for each extract, 30 μg protein was loaded and separated alongside protein standards. The separated proteins were then transferred onto a nitrocellulose membrane. Following transfer, the membranes were blocked with 5% fat-free milk powder in PBS and probed overnight at 4°C with primary antibodies in 5% bovine serum albumin at a 1:1,500 dilution.
The primary antibodies used for 0.5 M NaCl extracts were FN1 (SC-56391, clone SPM246, Santa Cruz Biotechnology Inc.), SPP1 (SC-20788, rabbit polyclonal, Santa Cruz Biotechnology Inc.), and fibromodulin (FMOD) (SC-25857, goat polyclonal, Santa Cruz Biotechnology Inc.). Primary antibodies for integrin beta 1 (ITB1) (SC-73610, clone 102DF5, Santa Cruz Biotechnology Inc.), CD68 (SC-70761, clone 3F103, Santa Cruz Biotechnology Inc.), CD31 (SC-31045, goat polyclonal, Santa Cruz Biotechnology Inc.), and SMA (A5691, clone 1A4, Sigma-Aldrich) were used on the SDS extracts. For human SMC experiments, the blots were also probed with antibodies to MMP9 (ab38898, rabbit polyclonal, Abcam), tenascin C (ab6393, clone BC-24, Abcam), fibronectin (SC-56391, clone SPM246, Santa Cruz Biotechnology Inc.), and LGALS3BP (70R-6085, rabbit polyclonal, Fitzgerald).
The membranes were then treated in appropriate horseradish peroxidase–conjugated secondary antibodies at a 1:2,000 dilution. Enhanced ChemiLuminescence (ECL, GE Healthcare) was used for imaging, and the exposed films were developed on a Xograph processor. ImageJ software (V.1.4.3.67; NIH) was used for densitometry of each lane of the developed films.

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