Transforming growth factor beta tgf β

Proteins present in hGMSC-CM were quantified by Bio-Rad Protein Assay Kit (Bio-Rad, USA). Proteins were separated using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, USA). After blotting, the PVDF membranes were stained with Ponceau S solution (SERVA, Denmark) to assess protein separation. Then, the membranes were incubated in blocking solution (5% skimmed milk in 1× PBS) at ambient temperature for 1 h. After blocking, the membranes were incubated with the following antibodies for overnight at 4°C: nerve growth factor (NGF) (1:250; Abcam, USA), NT3 (1:250; Santa Cruz Biotechnology Inc., USA), IL-10 (1:250; Santa Cruz Biotechnology Inc., USA), and transforming growth factor beta (TGF-β) (1:500; Abcam, USA). Subsequently, membranes were incubated with appropriate HRP-conjugated secondary antibodies IgG (1:2000; Santa Cruz Biotechnology Inc., USA). Finally, the membranes were subjected for protein detection using chemiluminescence (enhanced) solution kit (Luminata Western HRP Substrates, Millipore, USA). Protein band images were obtained using imaging software ChemiDoc™ MP (Bio-Rad, USA). Representative blot images from three separate analyses are given.