Cell culture supernatants were collected at 48 h. Vivaspin tubes (Sartorius Stedim Biotech GmbH, Goettingen, Germany) were used to collect proteins from the supernatant that had a higher molecular weight than 30 kDa. Protein concentrations were measured with a non-interfering protein assay kit (Calbiochem, EMD Bioscience Inc., Darmstadt, Germany) using a bovine serum albumin (BSA) standard curve (Bio-Rad Laboratories, Munich, Germany)
An anti-MMP-1 antibody produced in mice was used (Sigma-Aldrich Saint Louis, USA) with a goat anti-mouse secondary IgG-HRP antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany). 40 μg protein from each sample was diluted with sample buffer, while ddH2O was added to a final volume of 35 μl. The membranes were blocked in blocking buffer at room temperature for at least 1 h and afterward incubated with primary antibody overnight at 4°C. The primary antibodies were diluted 1:200 in blocking buffer. The membranes were incubated with diluted 1:5000 HRP-conjugated secondary antibodies for 1h at room temperature the following day. Images were analyzed using image reader LAS-R software (Leica Microsystems, Germany). IOD (integrated optical density) values were generated/analyzed with Gel-pro analyzer software (Media Cybernetics USA).
An anti-MMP-1 antibody produced in mice was used (Sigma-Aldrich Saint Louis, USA) with a goat anti-mouse secondary IgG-HRP antibody (Santa Cruz Biotechnology Inc., Heidelberg, Germany). 40 μg protein from each sample was diluted with sample buffer, while ddH2O was added to a final volume of 35 μl. The membranes were blocked in blocking buffer at room temperature for at least 1 h and afterward incubated with primary antibody overnight at 4°C. The primary antibodies were diluted 1:200 in blocking buffer. The membranes were incubated with diluted 1:5000 HRP-conjugated secondary antibodies for 1h at room temperature the following day. Images were analyzed using image reader LAS-R software (Leica Microsystems, Germany). IOD (integrated optical density) values were generated/analyzed with Gel-pro analyzer software (Media Cybernetics USA).