Anti-SMAD1/5/9 antibody (ab66737)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Rabbit (1:1000) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Cells were lysed in ice-cold Triton X-100 buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% Triton X-100, 1 mM phenylmethanesulfonylfluoride, PMSF) or RIPA buffer (20 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF) containing Complete Protease Inhibitor Cocktail (Roche, 11697498001). Cell lysates were spun down at 12,000 rpm for 10 min. In total, 50 μg of supernatants were resolved on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and blotted with the indicated antibodies. Primary antibodies used were: rabbit anti-WDR81 (1:500, Proteintech, 24874-1-AP), rabbit anti-p27 (1:100, Cell Signaling, 3688 S), rabbit anti-p57 (1:1000, Ruiying Biological, RLT3492), rabbit anti-p21 (1:1000, Santa Cruz, SC-397), rabbit anti-cyclinD1 (1:1000, Cell Signaling, 2922 S), rabbit anti-CDK4 (1:100, Santa Cruz, SC-260), rabbit anti-Smad2 (1:1000, Cell Signaling, 5339 S), rabbit anti-pSmad2 (1:1000, Cell Signaling, 3101 S), rabbit anti-pSmad1/5/9 (1:1000, Cell Signaling, 13820 S), rabbit anti-Smad1/5/9 (1:1000, Abcam, ab66737), rabbit anti-SARA (1:1000, Proteintech, 14821-1-AP), rabbit anti-TGFbetaRII (1:1000, Cell Signaling, 11888 S), rabbit anti-SARA (1:1000, Proteintech, 14821-1-AP), rabbit anti-VPS15 (1:1000, Abcam, ab128903), rabbit anti-VPS34 (1:1000, Cell Signaling, 3811 S), rabbit anti-Beclin1 (1:1000, MBL, PD017), rabbit anti-p-AKT (1:1000, Cell Signaling, 9018 S), rabbit anti-AKT (1:1000, Epitomics, 1085 S) and mouse anti-β-actin (1:1000, Sigma, A5441). The β-actin was used as the loading control.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-SMAD1/5/9 antibody (ab66737) below.

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