Anti runx2 antibody

Cells were lysed and extracted using lysis and extraction buffer (Pierce IP Lysis Buffer; cat. no. 87787; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols on day 7(24 (link)). Protein in the whole-cell lysates was quantified using the bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). A total of 10 µg/lane of protein samples were loaded on a 7.5% gel for collagen I and loaded on a 10% gel for RUNX2 and GAPDH experiments, respectively and then transferred to polyvinylidene difluoride membranes (Immun-Blot^®; Bio-Rad Laboratories, Inc.) for immunoblotting. The membranes were blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4˚C: Anti-collagen I (1:500; cat. no. ab6308; Abcam), anti-RUNX2 antibody (1:200; cat. no. ab76956; Abcam) and anti-GAPDH antibody (1:2,000; cat. no. ab9485; Abcam). After washing with TBS-0.1% Tween-20, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies, goat anti-mouse immunoglobulin G (IgG; cat. no. ab205719; Abcam) and goat anti-rabbit IgG (cat. no. ab205718; Abcam) at 1:10,000 dilution for 2 h at room temperature. The immunoblot signals were visualized using horseradish peroxidase substrate (cat. no. WBKLS0100; Merck KGaA).

To detect the osteogenic-related protein expression in the MSCs in each group, cells were incubated in an osteogenic induction medium for 1 week. Total protein was then extracted with cell lysis buffer and measured with a BCA kit (Thermo Scientific). Then, 40 μg total protein was separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels and transferred onto polyvinylidene difluoride membranes; after blocking with 5% non-fat dry milk, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-Runx2 antibody (Abcam, Cambridge, CA, USA), anti-Collagen I antibody (CST, Beverly, MA, USA), and anti-GAPDH antibody (CST, Beverly, MA, USA). All the antibodies were rabbit monoclonals and were diluted 1:1000 upon use. After rinsing with PBS, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit antibody (CST, Beverly, MA, USA) at 1:2000 dilution for 1 h at 37°C. After rinsing again, the membrane was scanned in an Odyssey Scanner (Li-COR Biosciences, Lincoln, NE, USA).

After 7 days of culture, cells were lysed, and the resulting protein-containing supernatants were transferred to PVDF (polyvinylidene difluoride) membranes. After blocking, the membranes were incubated with anti-Runx2 antibody (1: 1000, Abcam, Cambridge, MA, USA) overnight, followed by incubation with an HRP-conjugated secondary antibody (1: 3000, Abcam) for 2 hours the next day. Targeted protein bands were visualized using an ECL kit (CWBIO, Beijing, China). The intensity of the protein bands was quantified by Adobe Photoshop software.
The general steps for immunofluorescence were described earlier. The primary antibody was an anti-Runx2 antibody (1: 200). The secondary antibody was a rhodamine-conjugated anti-rabbit antibody (1: 200, Invitrogen, Carlsbad, CA, USA). The images were analyzed by Image-Pro Plus software.