Sc 11769

Paraffin sections in 5 µm were used. Osteoclast was carried out using a standard protocol by TRAP (tartrate-resistant acid phosphatase) staining (Sigma–Aldrich). Immunohistochemical staining was achieved by applying a standard protocol with the R&D HRP-DAB staining kit (R&D Systems, USA). Primary antibodies matrix metallopeptidase 13 (MMP13, Abcam, Cambridge, UK; 1:200, ab3208), osterix (OSX) (Abcam, 1:200, ab22552), osteocalcin (OCN) (Takara Bio Inc., Shiga, Japan; 1:200, M137); a TGFβ pathway-specific antibody against p-Smad2/3 (Santa Cruz Biotechnology, 1:100, sc-11769); and antibody against PTH1R (Abcam, 1:200, ab15750). A biotinylated secondary antibody was applied, and then all sections were counterstained with hematoxylin. Image J software evaluated the numbers of positive cells.

Frozen colon samples were homogenized in PBS with 0.1% protease inhibitor cocktail (Sigma) and phosphatase inhibitor cocktail (Sigma). Homogenates were centrifuged (12 000 g, 15 min, 4°C) and supernatants were collected and stored at −80°C. Protein concentration was determined following Bradford's colorimetric method. Aliquots of supernatants containing equal amounts of protein (25 µg) were separated on 4–12% NuPAGE gel (Invitrogen) and then transferred electrophoretically to a nitrocellulose membrane (Hybond, GE Healthcare, UK). After blocking in 5% nonfat dry milk, membranes were incubated with specific primary antibodies. Primary antibodies were obtained from Epitomics (Burlingame, CA, USA) for α-SMA (1184-1), from Santa Cruz biotechnology (Tebu, Le Perray-en-Yvelines, France) for p-Smad2/3 (sc-11769), from Abcam (Cambridge, UK) for COX-2 (sc-1747) and from Sigma for β-actin. They were incubated at a dilution of 1/1000 (p-Smad2/3), 1/2000 (α-SMA), 1/250 (COX-2,) and 1/5000 (β-actin). After three washes, filter was then incubated with secondary horseradish peroxidase linked anti-rabbit IgG for p-Smad2/3, α-SMA antibody, anti-goat IgG for COX-2 and anti-mouse IgG for β-actin. To check equal loading, the blots were analyzed for β-actin expression.

For pSmad3 blots, to confirm TGF-β inhibitors performed as expected, cells were serum starved for 1 hour in GMEM then placed in fibroblast media containing 10 ng/ml TGF-β (R&D Systems) with or without TGF-β inhibitors, then western blotting was performed as for all experiments, as follows. Cells were harvested in Trypsin-EDTA, lysed with 1x Nupage LDS lysis buffer (Life Technologies), with or without phosphatase inhibitors (HALT, Life Technologies), heated to 95°C for 10 minutes followed by sonication of 3 cycles of 15 s on the Misonix XL2000 sonicator on setting 2. Protein concentration was measured by BCA assay (Life Technologies), and 1-100 μg lysate was mixed with DTT (final 100 μM), then run at 150 V and transferred at 50 V for 3-hours with BioRad Mini-PROTEAN and Mini Trans-Blot Cell tanks respectively. Antibodies used included pSMAD2/3 antibody (Santa Cruz sc-11769), and B-ACTIN-HRP antibody (Abcam, ab20272l).