Bca quantitative method

The RIPA buffer containing protease inhibitors (Beyotime, Shanghai, China) was used to extract protein samples from target cells. The BCA quantitative method (Beyotime) was used to quantify the protein sample concentration. SDS-PAGE (10%; Invitrogen) was used to separate collected protein samples, which were then transferred to PVDF membranes (Millipore, Burlington, MA, USA). BSA (5%) was used to incubate the membranes for 2 h to prevent non-specific bindings. The following antibodies were used to incubate the membranes at 4°C overnight: ALP (ab67228, Abcam), OCN (ab93876, Abcam), RUNX2 (ab76956, Abcam), Osterix (ab209484, Abcam), DLX3 (ab178428, Abcam). Proper secondary antibodies (Cowin Biotech Co, Beijing, China) were used to incubate the membranes at room temperature for 2 h after the primary antibody incubation. Enhanced Chemiluminescence (ECL) Fluorescence Detection Kit (BB-3501; Amersham Pharmacia, Piscataway, NJ, USA) was used to visualize the blot signal on a Bio-Rad image analysis system (Bio-Rad, Hercules, CA, USA). The relative protein content is expressed by the gray value of the corresponding protein band/GAPDH protein band.

RIPA buffer (Catalog number: P0013C; Beyotime) and protease inhibitors were used to obtain total protein from either target tissues or transfected cells or untransfected target cells. The protein concentration was established by the BCA quantitative method (Catalog number: P0012, Beyotime). Then the protein samples were loaded into SDS‐PAGE (8%–15%) for separation and transferred to PVDF membrane. By incubating the membrane with a 5% milk blocking solution for 1 h, non‐specific binding prevention could be achieved. Then the membrane was incubated with anti‐CERCAM (1:500, 16411–1‐AP; Proteintech), anti‐PCNA (1:2000, 10205–2‐AP, Proteintech), anti‐Vimentin (1:2000, 10366–1‐AP, Proteintech), anti‐Twist (1:500, CSB‐PA025358LA01HU; Cusabio), anti‐N‐cadherin (1:2000, 22018–1‐AP, Proteintech), anti‐E‐cadherin (1:5000, 20874–1‐AP, Proteintech), anti‐Akt (Y409094, Abm), anti‐p‐Akt (1:500, Y011054, Phospho‐ser473; Abm), or anti‐β‐actin (1:5000, 60008–1‐Ig, Proteintech) at 4°C for 24 h, and then incubated with the matching secondary antibody at 37°C for 1 h. Enhanced chemiluminescence (ECL) reagents and Automatic chemiluminescence imaging system (Tanon 5200) were used to visualize proteins. Experiments were repeated for 3 times.