Ab133288

B cells were isolated from splenocytes from naïve mice as described above, and then the purity was assessed by flow cytometry. Purified B cells (>95%) were lysed with Pierce IP lysis buffer (Thermo Fisher Scientific, 87787). Cell lysates were then quantified and Western blots were performed exactly as previously described (23 (link)). The expression of EWSR1 and β-actin (as a loading control) was detected by using the primary antibodies rabbit anti-EWSR1 at 1:10,000 (Abcam, ab133288) and mouse anti–β-actin at 1:1,000 (Santa Cruz Biotechnology, sc-47778), followed by the secondary antibodies horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG at 1:5,000 (SouthernBiotech, 4050-05) and goat anti-mouse IgG at 1:5,000 (SouthernBiotech, 1010-05), respectively.

The tissues were homogenized and the supernatant was obtained following centrifugation (800 × g, 10 min) at room temperature. The protein concentration was measured using the BCA kit (Beijing Solarbio Science & Technology Co., Ltd.). A total of 40 µg protein sample was mixed with 10% SDS gel buffer and the protein was denatured by heating at 95°C for 5 min. Subsequently, the proteins were subjected to 12% SDS-PAGE and transferred to PVDF membranes, which were blocked with TBST solution containing 5% skimmed milk powder for 1 h at 4°C. The following antibodies were used: Rabbit anti-rat osteoprotegerin (OPG; 1:300, ab203061; Abcam), TRAP (1:10,000, ab133288; Abcam), dickkopf1 (DKK1; 1:2,000, ab61275; Abcam), Wnt2 (1:1,000, ab27794; Abcam), β-catenin (1:8,000, ab32572, Abcam) and β-actin (1:2,000, ab61275; Abcam). The polyclonal antibodies were diluted with TBST solution containing 3% bovine serum protein and incubated overnight at 4°C. Goat anti-rabbit IgG (1:1,000, ABIN 101988; antibodies-online, Aachen) labeled with horseradish peroxidase was incubated at room temperature for 1 h following pre-incubation. The PVDF membrane was incubated with the ECL substrate for 3-5 min following washing. The protein expression levels were normalized with β-actin and the analysis was performed using ImageJ 6.0 (NIH) software.