Ab119542

Total protein and nuclear protein lysates were extracted from cultured VICs using commercial buffers (Thermo Fisher, USA) according to the manufacturers' instructions. The primary antibodies used in this study were anti-Runx2 (ab23981, 1:500, Abcam, USA) and anti-IL-21 (ab119542, 1:1000, Abcam) along with anti-STAT3 (#4695, 1:1000, Cell Signaling Technology, USA) and anti-ALP (#8480, 1:1000 Cell Signaling Technology). Anti- GAPDH (60004-1-Ig, 1:5000) was purchased from Proteintech (China). Primary antibodies were incubated overnight at 4 °C. Blots were washed three times for 15 min with Tris-buffered saline with 0.1% Tween-20 (TBST) followed by incubation for 2 h at room temperature with secondary antibodies. After three washes for 15 minutes with TBST, specific staining was detected using chemiluminescence (ECL) system. All bands were densitometrically analysed with ImageJ.

Serum and culture supernatant for further researches were gathered and stored at −20°C. Commercial ELISA kit was used to measure the concentrations of CXCL13 (Abcam, USA, ab269370) and IL-21 (Abcam, USA, ab119542) according to the instructions from the manufacturer. The absorbance at 450 nm wavelength was read with the spectrophotometer (Eppendorf, Germany).