Nupage tris acetate pre cast gels

Changes in gene expression identified by qRT-PCR were confirmed by performing Western blotting on the cellular lysates with replicates. Briefly, endothelial tissue from portal veins (n=3) and hepatic veins (n=3) were homogenized in cell lysis buffer, and equal amounts of protein separated by 3–8% NuPAGE Tris-Acetate precast gels (Invitrogen, Carlsbad, CA). Proteins were then transferred to Immobilon-FL PVDF (Millipore, Billerica, MA) at 30V overnight in the cold room. Membranes were probed with anti-von Willebrand Factor (vWF) antibody and anti-endothelial protein C receptor (EPRC) antibody (Catalog #ab6994 and #ab78280, respectively, Abcam, Cambridge, MA) with GAPDH (Cell Signaling, Catalog # 2118, Beverly, MA) used as a loading control. Reliable porcine antibodies for proteins associated with PLAT and THBD genes were not commercially available at the time of the study. Primary antibodies were detected with infrared dye IR Dye 800CW or 680 RD-conjugated IgG (LI-COR Biosciences, Lincoln, NE). Immunoblots were imaged and quantified with the Odyssey Fc Dual-Mode Imaging System (LI-COR Biosciences).

Cell lysates containing 20 μg protein were separated on NuPAGE Tris-Acetate Pre-Cast Gels (Invitrogen) under denaturing conditions and the proteins were subsequently transferred to PVDF membranes (Bio-Rad) by electroblotting. The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: 1:1000; anti-rabbit LH2, # 21214-1-AP (Proteintech, Rosemont, IL, USA) and 1:200; anti-rabbit GAPDH #sc-25778 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing the membranes with 0.1% Tween-20 in Tris-buffered saline (TBS-T) three times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated IgG as a secondary antibody (Promega, Madison, WI, USA) at room temperature. Finally, the membranes were visualized using a ChemiDoc XRS Plus System (Bio-Rad, Hercules, CA, USA) with Image Lab software (Bio-Rad).

Cultured NRCMs were collected and extracted with RIPA buffer. An equal amount of total protein, determined with the Bio-Rad Coomassie Protein Assay (Bio-Rad Laboratories, Hercules, CA), was loaded to each lane. NuPAGE® Tris-Acetate Pre-Cast gels (Invitrogen, Grand Island, NY) were used to separate high molecular weight proteins over 200 kD. Proteins smaller than 200kD were separated by 4–12% NuPAGE® Bis-Tris Pre-Cast gels or 12% SDS-polyacrylamide gels. Target proteins were detected with specific antibodies using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo, Rockford, IL). Protein band intensity was quantified with NIH Image J software (http://rsb.info.nih.gov/ij/) using relative densitometric values on the duplicates of three independent experiments from each group.