Sds polyacrylamide gel

Total protein was isolated from BMSCs cultured under NPWT or normal condition or from wound tissues harvested at the indicated time points with a Total Protein Extraction Kit (Aspen, China). Equal amounts of protein from cells or tissue lysates were loaded onto a 5% SDS polyacrylamide gel (Aspen, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Membranes were blocked with 5% BSA in TBS and then incubated with primary antibodies against CD31 (1 : 500), α-SMA (1 : 5000), VEGF (1 : 1000), NG2 (1 : 100), and β-actin (1 : 10,000 for cell lysates and 1 : 3000 for tissue lysates) for overnight at 4°C. Next, an HRP-conjugated secondary antibody was applied (1 : 10,000) and detected with the Immobilon Western Chemiluminescent HRP Substrate system (Millipore, USA). All of the antibodies were purchased from Abcam Inc (United Kingdom).

Total protein was isolated from chondrocytes or harvested specimens with a Total Protein Extraction Kit (Aspen, China). Equal amounts of protein from cell or tissue lysates were loaded onto a 5% SDS polyacrylamide gel (Aspen, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Membranes were blocked with 5% BSA in TBS and then incubated with primary antibodies against IL-1β (1 : 500), TNF-α (1 : 1000), MMP-13 (1 : 500), p38 (1 : 2000), p-p38 (1 : 1000), and β-actin (1 : 10000) overnight at 4°C. Next, an HRP-conjugated secondary antibody was applied (1 : 10000) and detected with the Immobilon Western Chemiluminescent HRP Substrate system (Millipore, USA). All of the antibodies were purchased from Abcam Inc. (United Kingdom).