Evaluating Hypoxia and HGF Effects on Protein Expression

Some flasks containing 1833 cells infected with miR-125b mimic were exposed to hypoxia^{50 (link)}, or to 100 ng/ml HGF under starvation^{9 (link)}, and total and nuclear protein extracts were prepared. To evaluate ETS1, HIF1A and PTGS2, immunoblots were performed with anti-Ets-1 (1:2000), anti-HIF-1α (clone54) (1:350, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), and anti-COX2 (1:100). Also, anti-E-cadherin (1:1000, clone 36 Transduction Laboratories, Bedford, MA, USA), anti-MMP2 (1:500, Novus Biologicals, Abingdon, UK), anti-vimentin (1:500 Santa-Cruz Biotechnology), anti-SPARC (1:200, H-90 Santa Cruz Biotechnology), anti-osteocalcin (5 μg/ml, Abcam, Cambridge, UK), anti-B23 (1:1000, H106 Santa Cruz Biotechnology), or anti-vinculin (1:1000, Cell Signaling, Leiden, The Netherlands) was used for immunoblotting. Densitometric analysis was performed after reaction with ECL plus chemiluminescence kit from Thermo-Fisher Scientific (Waltham, MA, USA) or Clarity Max Western ECL Substrate-Luminol Solution (Biorad, Hercules, CA, USA).