Bmpr1a

Cell or the whole kidney tissue lysates were extracted in SDS lysis buffer containing 1 × PMSF (Aidlab, Beijing, China) and 1 × phosphatase inhibitor (Aidlab, Beijing, China). Western blots were performed as indicated with the following primary antibodies: Mtdh, fibronectin, BMPR1A, and Smad1/5/9 (Abcam, Cambridge, Britain); Col I (Boster, Wuhan, China); α-SMA (Sigma-Aldrich, Missouri, USA); E-cadherin (BD Biosciences, California, USA); p-P38 MAPK, P38 MAPK, p-ERK, ERK, and p-Smad1/5/9 (Cell Signalling Technology, Massachusetts, USA), and GAPDH (EarthOx Life Science, California, USA). The blots were subsequently incubated with an HRP-conjugated secondary antibody (EarthOx Life Science, California, USA) for 1–2 h at ambient temperature. Then, the membranes with immobilized antibodies were detected by enhanced chemiluminescence (Immobilon^TM Western, Millipore, Massachusetts, USA). Quantification was performed by measuring the grey scale intensity of the bands using Photoshop CS5 software (Adobe System Inc., California, USA).

Immunochemistry and immunofluorescence were performed as described previously [44 (link)]. Skin samples were fixed in 4% PFA, paraffin-embedded 5-μm sections were stained with hematoxylin and eosin (H&E). For immunofluorescence staining, paraffin sections were microwave pretreated, and incubated with primary antibodies, then incubated with secondary antibodies (Invitrogen) and counterstained with DAPI in mounting media. The following antibodies and dilutions were used: K14 (rabbit, 1:1000, Covance), K14 (mouse, 1:500, Abcam),K5 (rabbit, 1:1000, Covance), Sox9 (rabbit, 1:100, Santa Cruz), AE13(mouse, 1:100, Abcam), AE15 (mouse, 1:100, Abcam), BrdU (rat, 1:200, Abcam), Cleaved Caspase-3 (rabbit, 1:100, Cell Signaling), Gata-3 (mouse, 1:100, Santa Cruz), K17 (rabbit, 1:100, Abcam), Foxn1 (mouse, 1:100, Santa Cruz), Dlx3 (rabbit, 1:100, Santa Cruz), Sostdc1 (rabbit, 1:100, Abcam), Hoxc13 (mouse, 1:100, Sigma), Lef1 (rabbit, 1:100, Cell Signaling), Ki67 (mouse, 1:100,Novacastra), Bmpr1a (rabbit, 1:100, Abcam), Msx2 (rabbit, 1:200, Santa Cruz), k15 (mouse, 1:150, Vector Labs), CD49f (rat,1:100,BD), p63 (mouse, 1:200, Santa Cruz), K10 (rabbit, 1:1000, Covance), Loricrin (rabbit, 1:500, Covance), K1 (rabbit, 1:500, Covance).

Cells or bone tissues were homogenized in T-PER reagent (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). 20 μg of protein lysates of each sample was subjected to SDS-PAGE (8–12%) and transferred onto a nitrocellulose membrane. Immunoblots were incubated with the specific primary antibodies overnight at 4 °C with gentle shaking. After being washed three times for 15 min each with TBST (Tris-buffered saline, 0.1% Tween 20), blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma) for one hour. Next, the protein level was detected with Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and visualized by FluorChem E system (Protein Simple, CA, USA). The following antibodies were used: p-Smad1/5/8 (Cell Signaling, 9511), Tak1 (Cell Signaling, 4505, Boston, MA), Bmpr1a (Abcam, ab38560), β-Actin (Santa Cruz, Sc-47778).