P smad1 5 8

The decalcified paraffin sections for IHC eliminated the endogenous peroxidase activity with 3% H₂O₂ and retrieved antigens with boiled citrate buffer. Then, the sections were treated with 3% bovine serum albumin and 10% normal goat or rabbit serum to reduce non-specific immunoreactions. The sections were incubated with rabbit polyclonal primary antibodies against p-Smad1/5/8, p-Erk1/2, p-Junk, p-p38, DSP from Santa Cruz (Santa Cruz Biotechnology, Inc., Dallas, TX, United States), and rabbit polyclonal primary antibodies against Shh, Gli1, Nfic, OSX from Abcam (Abcam, Cambridge, MA, United States) overnight at 4°C and then the biotinylated-conjugated secondary antibodies (goat anti-rabbit antibodies) at room temperature for 1 h. The immunopositive loci were detected by the ABC kit and the DAB kit (Vector Laboratories, Burlingame, CA, United States) following the manufacturer’s instructions.

Tibial bone homogenates were lysed in lysis buffer containing protease inhibitor (Roche, Mannheim, Germany) and centrifuged at 10,000× g for 10 min at 4 °C. The total protein levels were determined using a Bio-Rad protein kit (Bio-Rad, Hercules, CA, USA). The proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto Immobilon-P transfer membranes (Millipore Co., Bedford, MA, USA), which were blocked with 5% bovine serum albumin prior to incubation with a specific primary antibody against BMP-2 (Abcam, Cambridge, UK), RUNX2 (Abcam), p-SMAD1/5/8 (Santa Cruz Biotechnology, Inc., Santa Cruz, TX, USA), Wnt3a (Abcam), osteocalcin (Abcam), COL-1 (Abcam), phosphorylated serine/threonine kinase (p-AKT) (Cell Signaling Technology, Danvers, MA, USA), p-ERK (Cell Signaling Technology), p38 (Cell Signaling Technology) and β-actin (Cell Signaling Technology) followed by goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP)-conjugated secondary antibody (Zymax, San Francisco, CA, USA). The antigen-antibody complexes were visualized by enhanced chemiluminescence. Densitometric analysis of the signal was performed using a C-DiGit Blot Scanner (Li-COR Inc., Lincoln, NE, USA). Relative expression was quantified using Image J (NIH, Bethesda, Rockville, MD, USA) and compared to β-actin.

Tissue sections were fixed in 4% paraformaldehyde and processed as previously described [18] (link). Immunofluorescence was performed in detail as previously described [19] (link). We used specific antibodies for pSMAD1/5/8 (Santa Cruz Biotechnology), total SMAD and ALK2 (both from Santa Cruz Biotechnology), ABCC6 (MRP6 antibody S-20, Santa Cruz Biotechnology), albumin (Bethyl Laboratories). The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich) [19] (link).

Tibias were homogenized in RIPA lysis buffer including protease inhibitor (Roche, Mannheim, Germany) and centrifuged at 10,000× g for 10 min at 4 °C. The total protein levels were determined using a Bio-Rad Protein Assay Kit. The proteins were subjected to SDS-PAGE and transferred onto Immobilon-P transfer membranes, which were blocked with bovine serum albumin (5%) prior to incubation with specific primary antibodies raised against BMP-2, Wnt3a, RUNX2, or COL-1 (Abcam); p-SMAD 1/5/8 (Santa Cruz, Texas, USA); or p-ERK, p-p38, p-JNK, or β-actin (Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with the appropriate secondary antibodies, either goat anti-rabbit IgG H&L (HRP) (Abcam) or goat anti-mouse IgG H&L (HRP) (Abcam). The antigen-antibody complexes were visualized using enhanced chemiluminescence. Densitometric analysis was performed using a C-DiGit Blot Scanner (Li-COR Biosciences, Lincoln, NE, USA).

For the western blot analysis, each protein extract (30 μg of total protein) was resolved by SDS-PAGE using 10% polyacrylamide gels and subsequently transferred to Immun-Blot^® PVDF membranes (Bio-Rad, Hercules, CA, USA). The primary antibodies and their dilution factors were as follows BST2, 1:1000 (Santa Cruz Biotechnology, Dallas, TX, USA); RUNX2, 1:1000 (Santa Cruz Biotechnology); SMAD1, 1:1000 (Abcam, Cambridge, UK); SMAD4, 1:500 (Abcam); SMAD5. 1:500 (Abcam); SMAD 8, 1:500 (Abcam); T-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology); p-SMAD1/5/8, 1:500 (Santa Cruz Biotechnology), and β-actin, 1:1000 (Sigma-Aldrich). Secondary antibodies were used at a 1:5000 dilution. The detection of protein bands was facilitated by an Enhanced Chemiluminescence Kit (Elpis Biotech) and membranes exposures to X-ray film (Amersham, Buckinghamshire, UK).