Bglap

Proteins in femurs, exosomes, or MC3T3-E1 cells were collected in RIPA buffer (Thermo Scientific, USA). The same amount of protein samples was loaded onto NuPage Bis–Tris polyacrylamide gels (Invitrogen, USA) and then proteins were transferred onto polyvinylidene difluoride membranes. After blocking in milk (5% w/v) for 4 h at room temperature, the membranes were subsequently incubated overnight at 4 °C with primary antibodies against GAPDH (1:5000; Proteintech, USA), Runx2 (1:1000; Cell Signaling Technology, USA), Bglap (1:500; Abcam, USA), Col1a1 (1:1000; Abcam, US), CD81 (1:1000; Abcam, USA), and TSG101 (1:1000; Abcam, USA). Next, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000; Jackson, USA), and the signals were visualized using SuperSignal West substrate (Thermo Fisher Scientific, USA). The density was measured and analyzed using ImageJ software.

Tissue sections or primary cells were fixed and blocked with 5% Donkey serum (Sigma-Aldrich, USA). Samples were then incubated with primary antibodies at 4°C overnight. The following antibodies were used for staining: Bglap (Abcam, USA, ab93876, 5 ug/ml); Tppp3 (Abcam, USA, ab150998, 1:50); Mkx (Lifespan Biosciences Inc., USA, LS-B8063, 1 ug/ml); Gli1 (R&D, USA, MAB3324, 10 ug/ml); Gli2 (R&D, USA, AF3635, 5 ug/ml); pH3 (Santa Cruz, USA, sc-8656-R, 1:200); Donkey anti-rabbit IgG (H + L), Alexa Fluor 594 conjugate (Thermo Fisher Scientific, USA, A21207, 1:500); Donkey anti-rat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A21208, 1:500); Donkey anti-goat IgG (H + L), Alexa Fluor 488 conjugate (Thermo Fisher Scientific, USA, A11055, 1:500); and Goat anti-rabbit IgG (H + L), Alexa Fluor 633 conjugate (Thermo Fisher Scientific, USA, A21072, 1:500). Samples were washed with PBS and incubated with secondary antibodies at room temperature for 1 hr. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, USA). Signals were detected under fluorescence microscopy. For TUNEL assay, procedures were followed as described by the manufacturer using the ApopTag Plus In Situ Apoptosis Fluorescein Detection Kit (Millipore, USA, s7111).

MC3T3-E1 cells were harvested into RIPA buffer (Thermo Scientific, USA). Equal amounts of protein samples were loaded onto NuPage Bis-Tris polyacrylamide gels (Invitrogen, USA). Next, the proteins were transferred onto polyvinylidene difluoride membranes. These membranes were then blocked with 5% milk for 4 h at room temperature and subsequently incubated at 4 °C overnight with primary antibodies for the following specific genes: Runx2 (1:1000; Cell Signaling Technology, USA), Bglap (1:500; Abcam, USA), Col1a1 (1:1000; Abcam, USA), ELK1 (1:500; Abcam, USA), pELK1 (1:1000; Abcam, USA), Bax (1:1000; Cell Signaling Technology, USA), caspase-3 (1:1000; Cell Signaling Technology, USA), Bcl-2 (1:1000; Cell Signaling Technology, USA), and GAPDH (1:5000; Proteintech, USA). Next, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:5000; Jackson, USA), and the signals were visualized using Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometry analyses were performed using ImageJ software (NIH).