Ab175385

Cells (1×10⁷) under 80% confluence or IVD tissues (0.1 g) were lysed in 1 mL ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher; #89901) containing the protease inhibitor (Abcam; #ab142778). Equal amounts (30 µg) of proteins were loaded into the wells of SDS-PAGE gel and separated by electrophoresis. Proteins were transferred onto the PVDF (polyvinylidene fluoride) membrane (Sigma-Aldrich; #IPSN07852) and blocked with 5% fat-free milk for one hour at room temperature. The membranes were then incubated with primary antibodies, including anti-BMPR1a (Abcam; #ab264043), anti-BMPR1b (Abcam; #ab175385), anti-BMPR2 (Abcam; #ab96826), anti-Smad1/5/8 (Sigma-Aldrich; #SAB2702532), anti-pSmad1/5/8 (Sigma-Aldrich; #AB3848-I), anti-Smad4 (Sigma-Aldrich; #HPA019154), anti-Puma (Abcam; #ab9645), anti-Apaf-1 (Abcam; #ab233786), anti-CASP9 (Abcam; #ab184786), anti-CASP3 (Thermo Fisher; #MA1-16843), anti-HDAC1 (Abcam; #ab7028), and anti-β-actin (Sigma-Aldrich; #A2066). After incubation at 4 °C overnight, membranes were washed 5 times with a PBS buffer containing 0.1% Tween-20 (Sigma-Aldrich; #P9416) and then probed with secondary antibodies (Abcam; #ab6721 and #ab6728). Protein signals were recorded by the Bio-Rad Gel Imaging System (Bio-Rad, Shanghai, China; #1708265).

A standard indirect IHC procedure was used to detect IGF-1, BMPR1b and MMP-10 expression (Dobie et al., 2015 (link)). Primary antibodies were anti-IGF-1 (Abcam, ab9572, 1:500 dilution), BMPR1b (Abcam, ab175385, 1:100 dilution) and MMP-10 (Abcam, ab199688, 1:100 dilution). Control sections were incubated with an equal amount of rabbit IgG in place of the primary antibody. Sections were counterstained by Haematoxylin, mounted and images captured using a Zeiss AxioImager brightfield microscope. Fixation and antigen retrieval were carried out using the same methodology throughout and all IHC for each antibody was performed on the same day under identical conditions, with control specimens also tested for each genotype. After the images were imported into Fiji, the Haematoxylin-DAB colour deconvolution plugin was used to separate the Haematoxylin and DAB components, and the ‘analyse-measure’ tool used to determine the absorbance in a consistent region of each sample, containing both GP and metaphyseal bone. Optical density (OD) was then calculated using the equation: OD=negative (base10)log of mean intensity of transmitted image/illumination (max intensity of image).
Maximum intensity was taken to be 255 for 8-bit images in Fiji (Ruifrok and Johnston, 2001 (link)).