Phospho-Paxillin (Tyr31) Polyclonal Antibody

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	-Rabbit (1:500)

Protocol tips
-Rabbit (1:500)

Publication protocol

Following DMSO or 30 µM 15d-PGJ2 treatment for 24 h at 37°C, CGTH W-2 thyroid carcinoma cells were washed with PBS and homogenized in lysis buffer for 10 min at 4°C (10 mM ethyleneglycoltetraaceticacid, 2 mM MgCl2, 60 mM piperazine-N,N'-bis(2-ethanesculforic acid), 25 mM 4-(2-hydroxethyl)-1-piperazineethanesulfonic acid, 0.15% Triton X-100, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, 1 mM NaF and 1 mM phenylmethylsulfonyl fluoride). Subsequently an equal volume of sample buffer (0.25% bromophenol blue, 0.5 M dithiothreitol, 50% glycerol, 10% SDS) was added and the mixture was heated at 90°C for 3 min. Proteins (60 µg per lane) were electrophoresed by 10% SDS-PAGE and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Strips from the membrane were blocked by incubating for 30 min at room temperature with 5% non-fat milk in TBS (pH 8.2) containing 0.1% Tween (TBST) and incubated overnight at 4°C with a 1:1,000 dilution of monoclonal mouse antibodies against integrin β1 (cat. no., 610467), FAK (cat. no., 610087), paxillin (cat. no., 610051), N-cad (cat. no., 610920), p120-ctn (cat. no., 610133; BD Biosciences), vinculin (cat. no., V4505) or β-actin (cat. no., A3854; Sigma-Aldrich; Merck Millipore), or a 1:500 dilution of rabbit antibodies against phosphorylated FAK (cat. no., 44–624G), phosphorylated paxillin (cat. no., 44-720G; Thermo Fisher Scientific, Inc.) and a 1:1,000 dilution of GAPDH (cat. no., 2251-1), Na+/K+ ATPase (cat. no., 2047-1; Epitomics, Burlingame, CA, USA) in TBST. Following TBST washes, the strips were incubated for 2 h at room temperature with 1:7,500 dilution of alkaline phosphatase-conjugated anti-mouse (cat no., S3721) or anti-rabbit immunoglobulin G antibodies (cat. no., S3731; Promega Corporation, Madison, WI, USA) and bound antibody was visualized using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich; Merck Millipore) as the chromogen. The density of the bands on the nitrocellulose membrane was quantified with densitometry using Gel Pro 3.1 (Media Cybernetics, Inc., Rockville, MD, USA). The density of the band in the control sample was taken as 100% and the density of the band in the test sample was expressed as a percentage of the control density. All the experiments were performed ≥3 times and the data are expressed as the mean ± standard deviation.

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