Anti psmad3 phospho s423 s425

Protein lysates were obtained from minced bones and from OBs using RIPA lysis buffer supplemented with protease inhibitors as previously described(47 (link)). Following protein quantitation using RC DC Protein Assay (BioRad), proteins were separated on 6% (collagen), 9% (pSMAD2 and SMAD2/3), 10% (TRIC-B, HSP47) or 15% (TGF-β) SDS-PAGE and transferred on PVDF membrane. The membranes were incubated o/n at 4°C with 1:1000 anti-TRIC-B (Invitrogen), anti-SMAD2/3 (Cell Signaling), anti-pSMAD2 (Abcam), anti-pSMAD3 (Abcam), anti-HSP47 (Santa Cruz) anti-collagen I (Abcam), anti-TGF-b (R&D System), anti-pCaMKII (Abcam) antibodies in TBS-T. ImageQuant LAS 4000 (GE Healthcare) and the ImageQuant LAS 4000 1.2 software were used for images acquisition. ImageQuant TL analysis software was employed for band intensity evaluation. At least biological triplicates were performed. For each gel, the expression of the mutant samples was expressed as fold difference compared to controls. Protein loading normalization was determined using anti-β-actin antibody (Santa Cruz Biotechnology) or total proteins staining by Swift^™ Membrane Stain (G-Biosciences). For human cells, the following antibodies and dilutions were used: anti-pSMAD3 (phospho S423/S425) 1:1000 (Rockland) and anti-GAPDH 1 :1000 (Cell Signaling).