Rabbit anti phd2

Proteins in the tumor and paired ANLTs in a set of 24 randomly selected cases (from 81 HCC patients) were extracted using RIPA lysis buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) with phenylmethanesulphonyl fluoride (17.4 μg/μl) and a protease inhibitor cocktail (0.89 μg/μl, Sigma-Aldrich). After the protein concentration was determined using a BCA Protein Assay Kit (Pierce), the proteins were separated on SDS-polyacrylamide gels followed by transfer to polyvinylidene difluoride membranes, which were blocked in 5% fat-free milk with TBS-Tween-20 at room temperature for 1 h, followed by incubation with primary antibodies mouse anti-PHD1 (1:1000; Abcam, Cambridge, UK), rabbit anti-PHD2 (1:1000; NOVUS Biologicals, Littleton CO, USA), rabbit anti-PHD3 (1:1000; NOVUS Biologicals, Littleton CO, USA), mouse anti-FIH (1:200; NOVUS Biologicals, Littleton CO, USA), or anti-β actin antibody (1:1000; Santa Cruz, USA) at 4°C overnight. Following incubation with an HRP-conjugated secondary antibody (1:5000; Jackson Immmuno Research Laboratories, INC, West Grove, PA, USA), the blots were visualized using enhanced chemiluminescence (Pierce Biotechnology, USA) and were exposed to the Syngene GBOX/iCHE gel imaging systems. The intensity of each band was analyzed by Image J and normalized to β-actin.