Bca protein assay

Pretreatment of antler chondrocytes was carried out with MLT (Meilunbio, Dalian, China) for 2 h, then VEGF was added for 24 h. The treated cells were exposed to lysis buffer (RIPA lysis and 50 × Cocktail) (Servicebio, Wuhan, China) for 20 min. Extract protein and detect protein concentration by the BCA protein assay (Servicebio, Wuhan, China). The total protein (20 μg) was separated by SDS-PAGE. Subsequently, the separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes and blocked with 5% skimmed milk powder in TBS for 2 h and incubated PVDF membrane with primary antibody at 4 °C overnight. The following antibodies were used: anti-Melatonin Receptor 1A (1:1000, GTX31054, GeneTex, San Antonin, TX, USA), anti-Col2a (1:2000, 15943, Proteintech, Wuhan, China), anti-YAP1 (1:1000, GTX129151, GeneTex), anti-p-AKT (Ser473) (1:1000, 4070, Cell Signaling Technology (CST) Biological reagents Company Ltd., Shanghai, China), anti-AKT (1:1000, C67E7, CST), anti-p-CREB(Ser133) (1:1000, 9198, CST) and anti-p-STAT5(Tyr694) (1:1000, 4322, CST), anti-STAT5 (1:1000, 25656, CST). After washing for three times by TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h. At last, the membranes were treated with ECL chemiluminescence reagent and exposed to X-ray film to observe protein bands.

After total protein extraction from iSMCs, the proteins were measured using the BCA protein assay (Servicebio). A Bis–Tris gel (ACE Biotechnology) was filled with fresh extracts. Proteins were then transferred to a polyvinylidene fluoride membrane and blocked with 5% BSA. An anti-ACTA2 antibody (1:3000, Abcam, ab7817) was used to test ACTA2 expression. Secondary antibodies coupled to horseradish peroxidase were used to detect the signals.