Ab186838

Humeral head and supraspinatus tendon composite were harvested and fixed in the 4% paraformaldehyde in PBS overnight at room temperature. After decalcified and dehydrated, samples were embedded in Tissue-Tek O.C.T. Compound (SAKURA, Torrance, USA) and cut into 10 μm thickness of sagittal sections. Cell samples were fixed in the 4% paraformaldehyde in PBS for 30 min at room temperature. Both the parts and cell samples were blocked in 5% BSA for 40 min at room temperature and incubated with the primary antibodies anti-DMP1 (Abcam, 1:400, ab13970), anti-GFP (Abcam, 1:400, ab13970 or 1:400, ab290), anti-Sox9 (Abcam, 1:400; ab185966), anti-TGF-βR2 (Abcam, 1:400, ab186838) at 4 °C overnight. After washing, the sections were then incubated with the respective secondary antibodies (1:500, Abcam) for 1 hr at room temperature and sealed with DAPI. The images were captured with a Leica TCS-SP8 confocal microscope (Leica, Germany).

Adipocyte lysates were prepared in a lysis buffer containing 50 mM HEPES (pH 7.4), 10 mM EDTA, 50 mM sodium pyrophosphate, 0.1 M sodium fluoride, 10 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM benzamidine and 1% Triton X-100. After protein electrophoresis and transfer, immunoblots were performed using rabbit anti-serum as primary antibody at a 1:1000 dilution. This dilution was used for each of the primary antibodies used for the present study. After washing, the blot was incubated with a 1:10,000 dilution of goat anti-rabbit horseradish peroxidase–conjugated secondary antibody followed by a chemiluminescent kit (Immobilon Western; EMD Millipore, Billerica, MA, USA) as previously described [56 (link)]. β-actin was used as a loading control. The maximum intensity of each band was quantified using ImageJ software. Ratios of TGF-β1, TGFBR1, TGFBR2, Smad3 and p-Smad3 were normalized to β-actin. Antibodies against TGF-β1 (ab9758). TGFBR1 (ab31013), TGFBR2 (ab186838), SMAD3 (ab227223) and p-Smad3 (ab118825) were products of Abcam (Cambridge, UK). Anti-rabbit IgG antisera were products of Cell Signaling Technology, Inc. (Danvers, MA, USA).

After lysing in RIPA buffer (Solarbio), cell lysates were centrifuged for protein collection. The concentration of protein sample was tested using a BCA kit (Abcam). Twenty microgram samples were loaded on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Solarbio). Next, the membranes were blocked in 5% non-fat milk, and then interacted with primary antibodies anti-Sry-type high-mobility-group box 9 (SOX9) (ab3697, 1:1000 dilution, Abcam), anti-collagen type II α 1 (COL2A1) (AB761, 1:100 dilution, Sigma–Aldrich), anti-Aggrecan (AB1031, 1:1000 dilution, Sigma–Aldrich), anti-TGFBR2 (ab186838, 1:500 dilution, Abcam) or anti-β-actin (ab227387, 1:5000 dilution, Abcam) and the secondary antibody conjugated by horseradish peroxidase (ab205718, 1:20000 dilution, Abcam). β-actin functioned as a loading control. Subsequently, the protein blots were developed via exposing to ECL reagent (Solarbio) and tested by ImageJ software (NIH, Bethesda, MD, U.S.A.). Relative protein level was normalized to the control group.