Nb 100122

Total RNA was extracted from CRC cells using the TRIzol® RNA purification reagent. RNA quantity and purity were determined by using a NanoDrop ND-1000. Total RNA (1 μg) from each sample was reverse transcribed and real-time RT-PCR measurements were performed as described previously [40 (link), 41 (link)] using a Mx3000P apparatus (Agilent) with the corresponding SYBR Green kit. PCR primers were designed with Primer 3 (Agilent) as follows: VEGF, upper, 5’-CGAAGTGGTGAAGTTCATGGATG-3’, lower, 5’-TTCTGTATCAGTC TTTCCTGGTGAG. 36B4 (also known as RPLP0), upper, 5’-GATTGGCTACCCAACTGTTG-3’; lower, 5’-CAGGGGCAGCAGCCAC AAA-3’. Gene expression was normalized to 36B4.
Western blot analysis was carried out as described previously [42 (link)]. The primary antibodies were directed against HIF-1alpha (BD Bioscience # 610958), HIF-2alpha (Novus Biological # NB-100122) or beta-actin (Santa Cruz # sc-47778), while the corresponding secondary antibodies were purchased from Jackson ImmunoResearch.

For immunoblotting cells were harvested in PBS (4 °C), cell pellets were lysed in 10 mM Tris/HCl (pH 7,5), 2% SDS, 2 mM EGTA, 20 mM NaF or in 50 mM Tris/HCl buffer (pH 7.5), 1% Triton X-100, 150 mM NaCl, 10 mM sodium pyrophosphate, 20 mM NaF, 1 mM sodium orthovanadate and 1% complete protease inhibitor cocktail (Roche). Protein (25 μg) lysates were subjected to SDS–PAGE and western blot analysis was performed using antibodies specific for HIF-1α (BD Transduction Laboratories, no. 610958), HIF-2α, PHD1, PHD2, PHD3 (Novus Biologicals, NB 100-122, NB-100-310, NB100-137 and NB-100-303, respectively), pEGFR (Tyr 1173; Santa Cruz Biotechnology), pEGFR (Tyr 1068; Cell Signalling), EGFR (Cell Signalling or Millipore) and tubulin as a loading control (Jackson Lab, DLN09992 or Invitrogen). Immunoreactive bands were detected with horse radish peroxidase (HRP)-conjugated secondary antibodies through enhanced chemiluminescence (ECL, Thermo, Perkin-Elmer and GE Healthcare).