Anti sclerostin

15 μg of protein from bone and muscle tissues were subjected to 12% SDS-PAGE and subsequently transferred to nitrocellulose membranes (Hybond, Amersham). The blots were probed using primary antibody anti-Sclerostin (Abcam), anti-OxPhos Complex IV subunit I (Invitrogen) and anti-MyHC II (Abcam) and IRDye-labeled secondary antibodies (680/800 CW) (LI-COR Biosciences). For immunodetection, the Odyssey infrared imaging system was utilized (LI-COR Corp., Lincoln, NE). All data were normalized to background and loading controls.

MLO-Y4 cells were subjected to HiSMF for 48 h; the RIPA Lysis Buffer (Beyotime) containing 1-mM Phenylmethanesulfonyl fluoride (PMSF; Beyotime) was used to lyse the cells. A commercial BCA Protein Assay Kit (Beyotime) was used to examine the protein content. The proteins were loaded into the SDS-PAGE gels and were separated by electrophoresis, and then transferred onto the nitrocellulose (NC) membrane. The NC membranes were blocked with 5% nonfat-dried milk and were then incubated in the specific primary antibody, including anti-GAPDH (Proteintech, Rosemont, IL, USA), anti-Connexin 43 (Cx43；BioWorld, Nanjing, Jiangsu, China), anti-Sclerostin (Abcam, Cambridge, Cambridgeshire, UK), anti-Ferritin Heavy Chain (FTH1; Cell Signaling Technology, Boston, MA, USA), anti-divalent metal transporter 1 (DMT1; Abcam), and anti-ferroportin 1 (FPN1/SLC40A1; Abcam) overnight at 4 °C. After using tris buffered saline tween (TBST) to wash three times, NC membranes were incubated by the species-specific secondary antibody conjugated to horseradish peroxidase (Kangwei Century, Beijing, China) for 1 h at indoor temperature. A ECL Plus Western Blotting Detection System (Tanon, Shanghai, China) was used to image the immunoreactive bands. The grayscale bands were determined by ImageJ 1.8.0 software (NIH, Bethesda, MD, USA).