Ab40771

For the western blot analysis, the antibodies used were as follows: E-cadherin (AF0131, 1:1000, Affinity), VEGFA (sc-57496, 1:200, Santa Cruz), SMAD5 (ab40771, 1:5000, Abcam), MMP1 (A0568, 1:1000, Boster), HIF1α (sc-13515, 1:200, Santa Cruz), Snail (3099-1-AP, 1:1000, Proteintech), and Twist1 (5465-1-AP, 1:1000, Proteintech), with GAPDH (AB0037, 1:5000, Abways) used as an internal control.

Total protein was extracted and quantified using radioimmunoprecipitation assay buffer and the BCA Kit (both from KeyGen; Nanjing, China), respectively. Equal amounts of protein were separated through electrophoresis on 10% SDS-PAGE gels. After transferring to polyvinylidene fluoride membranes and blocking with 5% nonfat milk, the membranes were incubated with primary antibodies for SMAD5 (ab40771) or GAPDH (ab128915; both from Abcam, Cambridge, MA, USA) at 4°C overnight. Incubation with horseradish peroxidase-coupled secondary antibodies (ab205718; Abcam) was performed, and the BeyoECL Moon Kit (Beyotime) was used to generate the chemiluminescent signal.

Samples were homogenized in 100 μL ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with a proteinase inhibitor cocktail. The blots were probed with the primary antibodies for E-cadherin (ab76055, Abcam, Cambridge, MA, USA), Vimentin (ab92547, Abcam), α-SMA (ab5694, Abcam), SMAD3 (ab40854, Abcam) and SMAD5 (ab40771, Abcam).

The expression of SMAD5 was detected by immunohistochemical streptavidin-perosidase (SP) kits (ab64261, Abcam Inc., Cambridge, UK). Briefly, NPC and adjacent normal tissues were fixed by 10% formalin, paraffin embedded, and then sectioned. The blocked sections were then incubated with primary antibody of rabbit monoclonal antibody to SMAD5 (1:50, ab40771, Abcam Inc., Cambridge, UK). Goat anti-rabbit immunoglobulin G (IgG) (1:500, ab97051, Abcam Inc., Cambridge, UK) was applied as secondary antibody. SMAD5 protein was predominantly expressed in the cytoplasm, and brown granules were visualized in the SMAD5-positive cells after staining. Images were captured and the positive rates were counted by Image J software.

The bone tissue was located at the fracture site, placed in liquid nitrogen, and ground; then, it was lysed using lysis buffer, and the total protein concentration of lysates was measured using a micro BCA protein assay kit (Pierce, Rockford, IL). The samples were separated on 12% SDS-polyacrylamide gels and then electrophoretically transferred to polyvinylidene difluoride membranes. After blocking in 5% non-fat milk solution for 1 h, the membranes were incubated with anti-Osterix (Abcam, ab209484, Cambridge, MA, dilution 1:1000), anti-BMP-2 (Abcam, ab14933, Cambridge, MA, dilution 1:500), anti-phosphorylated-Smad5 (Abcam, ab92698, Cambridge, MA, dilution 1:200), anti-Smad5 (Abcam, ab40771, Cambridge, MA, dilution 1:1000), anti-Runx2 (Abcam, ab76956, Cambridge, MA, dilution 1:200) and GAPDH antibodies for 2 h at 37 °C. Then, the membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Next, the membranes were treated with ECL solution (Millipore, Darmstadt, Germany) and then imaged using ImageJ (Bethesda, MD). The grey values of each protein band were measured by Photoshop CS5 software (Adobe, San Jose, CA). The relative band intensity was assessed as the ratio of the grey value of each protein to that of the corresponding GAPDH.