Smad2 3 d7g7 xp rabbit mab

Cells were washed with ice-cold 0.01 M PBS, lysed in the RIPA buffer with protease inhibitor and protein phosphatase inhibitor for 30 min on ice. Then cells were scraped and centrifuged with 12,000 rcf for 15 min. Protein concentration was determined with BCA Protein assay kit (Thermo Scientific, Rockford, IL, USA). The proteins were separated by SDS-PAGE and transferred onto PVDF membrane. The primary antibodies used were Human FSTL1 Antibody (1:200, AF1694; R&D Systems, Minneapolis, MN, USA), GAPDH (1:3,000, #5174; Cell Signaling Technology Inc., Beverly, MA, USA), Smad2/3 (D7G7) XP^® Rabbit mAb (1:1,000, #8685; Cell Signaling Technology Inc.), anti-phospho-Smad2/3 (pThr8) (1:1,000, SAB4504208; Sigma-Aldrich), anti-Smad1/5/8 antibody (1:200, sc-6031-R; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-p-Smad1/5/8 (Ser 463/Ser 465) antibody (1:200, sc-12353; Santa Cruz Biotechnology); anti-Smad3 antibody (1:1,000, ab28379; Abcam, Cambridge, MA, USA); anti-Smad3 (phospho S423+S425) antibody [EP823Y] (1:2,000, ab52903; Abcam); caspase-3 antibody (1:1,000, #9662; Cell Signaling Technology Inc.); cleaved caspase-3 (Asp175) (5A1E) (1:1,000, #9664; Cell Signaling Technology Inc.).