Immobilon fl pvdf

Western blots were performed with protein samples obtained from the same transfected cells for which total RNAs were analyzed by Northern blot (see above). Thirty micrograms of total protein were separated by electrophoresis. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL PVDF, Millipore, Billerica, MA). The proteins of interest were detected with the following antibodies: anti-T7 Epitope Tag antibody (Millipore), TAP Tag polyclonal antibody (Thermo Scientific), FLAG-M2 antibody (Agilent Technologies, Santa Clara, CA), monoclonal anti-α-Tubulin (Sigma), eIF3 p110 (B-6) antibody (Santa Cruz Biotechnology, Dallas, TX), anti-Mouse IRDye 680LT (LI-COR Biosciences, Lincoln, NE) and anti-Rabbit IRDye 800CW (LI-COR Biosciences). Fluorescent secondary antibody signals were detected with an Odyssey CLx (LI-COR Biosciences) and quantified with Image Studio software (LI-COR Biosciences). Detailed protocols are in the Supplemental Methods.

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).

Whole cell lysate was prepared by adding 5μL/mg tissue of ice cold lysis buffer (20mM HEPES, 1% Triton x-100, 1% sodium deoxycolate, 1mM DTT, 1% SDS, 1X protease inhibitors (Thermo Scientific Pierce Protein Biology), 1 mM NaF, 5 mM EDTA, 10 mM β-glycerophosphate and 250 μM Na₃VO₄) and sonicated 3 times for 5 seconds on ice, clarified by centrifugation at 15,000 × g for 12 minutes at 4°C and stored at −80°C until use. Protein concentration was measured using detergent compatible protein quantification kit (Biorad) and final loading samples were prepared at 4μg/μL protein in LDS Sample Buffer (Life Technologies) with 50 mM DTT (Life Technologies). Sixty μg of whole cell lysate were loaded into each well of a NuPAGE NOVEX 4–12% Bis-Tris gel with MES running buffer, transferred onto Immobilon-FL PVDF (Millipore). A solution of 5% milk in TBS + 0.1% Tween 20 was used to block and dilute primary and secondary antibodies (1:5000, LiCor Biosciences, Rabbit antibody conjugated with IRdye 800, mouse antibody conjugated with IRdye 680), scans and quantifications were used using a LiCor Odyssey scanner. The primary antibodies included: ADAM10 (Rb 1:1000, Abcam ab1997), GFAP (Rb 1:50,000, Abcam ab7260), Actin (Ms 1:2000, Abcam ab3280), BACE1 (Rb 1:1000, Abcam ab2077).