Anti p smad1 5

The following antibodies were used in this study: anti-CD11b-FITC (Biolegend, 101206, dilution ratio: 1:100 for flow cytometry and immunofluorescence); anti-Ly-6G-PE (eBioscience, 12-9668-82, dilution ratio: 1:100 for flow cytometry); anti-Ly-6C-PE (Cell eBioscience, 12-5932-82, dilution ratio: 1:100 for flow cytometry); anti-p-Smad1/5 (Cell Signaling Technology, 9516, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-Smad1/5 (Cell Signaling Technology, 6,944, dilution ratio: 1:100 for immunofluorescence and 1:1000 for Western blot); anti-p-Stat3 (Cell Signaling Technology, 9,145, dilution ratio: 1:1000 for Western blot); anti-Stat3 (Cell Signaling Technology, 9,139, dilution ratio: 1:1000 for Western blot); anti-p-Erk (Cell Signaling Technology, 4,370, dilution ratio: 1:1000 for Western blot); anti-Erk (Cell Signaling Technology, 4,695, dilution ratio: 1:1000 for Western blot); anti-mouse IL6 (Biolegend, 504501, dilution ratio: 1:500 for neutralization); anti-S100A9 (Abcam, GB111149, dilution ratio: 1:2000 for IHC); anti-Ki67 (Servicebio, GB13030-2, dilution ratio: 1:500 for IHC), and anti-β-actin (Sigma, A1978, dilution ratio: 1:1000 for Western blot). Rat tail collagen I (Corning, 356236) and RhBMP2 (R&D, 355-BM-100) were also used. Tris–HCl, NaCl, and other chemicals were from Sigma.

Fresh intestinal tissues were washed in 1 mL of ice-cold PBS several times. Tissue and frozen steel beads were placed into a 2 mL centrifuge tube, and tissues were homogenized and lysed in lysis buffer with a protease and phosphatase inhibitor cocktail (Sigma). After clearing by centrifugation, protein concentrations in the supernatant were determined using a spectrophotometer (Thermo Fisher). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Significant results are shown, and similar results were obtained in at least three independent experiments. The primary antibodies were anti-β-catenin (BD Biosciences, lot 610154), anti-active β-catenin (Cell Signaling, lot D13A1), anti-psmad1/5 (Cell Signaling, lot 41D10); and anti-math1 (Santa Cruz, lot sc-136173). Proteins were detected by ECL WB detection regent (Thermo Fisher).

Immunoblot assay was performed as described previously^{60 (link)}. Briefly, proteins extracted in lysis buffer were separated by SDS–polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies overnight at 4 °C, and then incubated with a horseradish peroxidase-coupled secondary antibody. Detection was performed using a LumiGLO chemiluminescent substrate system. The following antibodies were used at indicated dilutions: anti-SHP2 (Santa Cruz Biotechnology, sc-7384), anti-SOX9 (Abcam, ab76997), anti-pSmad1/5 (Cell Signaling Technology, 9516), anti-p-Erk1/2 (Cell Signaling Technology, 4370), anti-ACTIN (Abmart, M20010).

Total protein was extracted with a whole cell lysis, and its concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific; No. 23225). Proteins were separated on 10%‐12% SDS‐polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore). The membranes were blocked with 5% bovine serum albumin and then incubated with the following primary antibodies: anti‐Runx2 (Cat No. 125561; Cell Signaling, 1:1000), anti‐GAPDH (Cat No. 5174; Cell Signaling, 1:1000), anti‐P‐Smad1/5 (Cat No. 9516; Cell Signaling, 1:1000), anti‐Smad1 (Cat No. 6944; Cell Signaling, 1:1000), OPN (Cat No. BS1264; Bioworld Technology, 1:1000), anti‐non‐p‐β‐catenin (Cat No. 19807; Cell Signaling, 1:1000), anti‐β‐catenin (Cat No. 8480; Cell Signaling, 1:1000), anti‐BMP2 (ab14933; Abcam, 1:600), anti‐Smad6 (ab13727; Abcam, 1:500), anti‐BMPR‐IA (sc5676; Santa, 1:200), anti‐BMPR‐IB (sc5679; Santa, 1:200) and anti‐BMPR‐II (sc393304; Santa, 1:200). The membranes were incubated with secondary antibodies, after washing 3 times for 10 minutes with TBST. Next, enhanced chemiluminescence reagent (Thermo Fisher Scientific) was used to detect the blots.

The hASCs were rinsed with ice PBS three times and immersed in RIPA buffer (HuaxingBio, Beijing, China) mixed with protease inhibitor cocktail (HuaxingBio). The pierce BCA protein assay kit (Thermo Fisher Scientific) was used to determine the protein concentration. A 25-μg sample of protein was added and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then followed by transfer to the polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Strips on the membranes were blocked with 5% nonfat dry milk (BioRuler, Danbury, CT, USA) for 1 h at room temperature, incubated overnight at 4 °C with primary antibodies at a dilution of 1:1000, and then for 1 h at room temperature with goat anti-rabbit secondary antibodies labeled with horseradish peroxidase (ZSGB-BIO, Beijing, China; ZB-2301) at a dilution of 1:10,000. The primary antibodies used were as follows: anti-GAPDH (ZSGB-BIO; TA-08), anti-NOTCH1 (Cell Signaling Technology, Beverly, MA, USA; 3608S), anti-HES1(Abcam, Cambridge, UK; ab108937), anti-LSD1 (Cell Signaling Technology; 2139S), anti-BMP2 (Abcam; ab14933), anti-SMAD4 (Abcam; ab40759), anti-phosphorylated SMAD1/5 (anti-p-SMAD1/5; Cell Signaling Technology; 9516S), and anti-RUNX2 (Cell Signaling Technology; 12556). Relative band intensities were measured with the ImageJ software.