Anti mouse igg antibody hrd

Cells were harvested in RIPA Lysis Buffer (strong) (CWbio, Taizhou, China; CW2333S) containing protease inhibitors (Roche, 4693124001). A total of 40 µg proteins were separated by 4%–20% gradient SDS-PAGE gels (GenScript, M42015C) and transferred to PVDF membranes (Millipore, Billerica, MA, USA; IPVH00010). The membranes were blocked at room temperature with 1% BSA for 1 h and incubated overnight at 4 °C with primary antibodies: rabbit Collagen I antibody (Cell Signaling Technology, 84336), mouse E-cadherin antibody (Abcam, ab1416), mouse α-SMA antibody (Sigma-Aldrich, A5228) and mouse β-actin antibody (Sigma-Aldrich, A1978). Then, the membranes were washed with TBST for 3 times and incubated for 1 h with a secondary antibody: anti-mouse IgG antibody (HRD) (Sigma-Aldrich, A9044) and anti-rabbit IgG antibody (HRD) (Sigma-Aldrich, A0545) at room temperature. Images were obtained using the ChemiDoc XRS + imaging system (Bio-Rad) and quantified using the Quantity One software.

RIPA Lysis Buffer (CWbio, China) containing protease inhibitors was used to collect samples (Roche, USA). Twenty grams of proteins were separated using 4%–20% gradient SDS‐PAGE gels (GenScript, USA) and transferred to PVDF membranes (Millipore, Germany). The membranes were blocked for 1 h at room temperature with 1% BSA before being incubated overnight at 4°C with primary antibodies: mouse anti‐RUNX2 antibody (Abcam, UK) at 1:1000, rabbit anti‐osteopontin (OPN) antibody (Affinity, USA) at 1:1000 and mouse anti‐GAPDH antibody (Proteintech, USA) at 1:10,000. Then, the membranes were washed with TBS and with Tween‐20 (TBST) three times, and incubated for 1 h with a secondary antibody: anti‐mouse IgG antibody (HRD) (Sigma‐Aldrich, USA) and anti‐rabbit IgG antibody (HRD) (Sigma‐Aldrich, USA) at room temperature. Images were obtained using the ChemiDoc XRS + imaging system (Bio‐Rad, USA).