Monoclonal rabbit antibody to β actin

S-Adenosyl-[methyl-3H]-L-methionine (3H-AdoMet) was purchased from PerkinElmer Life Sciences. A rabbit polyclonal antibody raised against full-length human recombinant CKB was purchased from Abnova (#PAB19125). A monoclonal rabbit antibody to β-actin (used as a loading control) was from Cell Signaling (#4970). Secondary antibody (horse radish peroxidase-linked donkey anti-rabbit IgG) and Pierce ECL Plus Western Blotting Substrate were purchased from GE/Amersham and Thermo/Pierce, respectively. Recombinant rat PIMT was expressed in E. coli and purified as described previously [9] (link). Recombinant human CKB was purchased from MyBioSource. Isoaspartyl delta-sleep inducing peptide (WAGGD∧ASGE; where ∧ designates an isopeptide bond) was purchased from Bachem.

Transfected Huh-7 cells were washed with ice-cold PBS and scraped from the wells in ice-cold buffer K (1 mM Tris–HCl, 1 mM EGTA, and 1 mM MgCl₂, pH 7.6) containing protease inhibitor cocktail (Sigma–Aldrich; catalog no.: P2714). Cells were manually lysed through 20 passes through a BD PrecisionGlide 25G needle, and the total protein concentrations were measured with a Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific; catalog no.: 23200). Proteins (25 μg) were resolved by SDS-PAGE (10%). A polyclonal rabbit primary antibody to human MTP (Abcam; catalog no.: ab63467) and a monoclonal rabbit antibody to β-actin (Cell Signaling Technology; catalog no.: 8457) were used at 1:1000 dilution. Anti-rabbit immunogobulin G, horseradish peroxidase–linked secondary antibody (Cell Signaling Technology; catalog no.: 7074) was used at 1:2000 dilution. The blots were developed with a ChemiDoc Touch Imaging system (Bio-Rad). To determine the MTP activity, 50 μg of total proteins was used. Fluorescently labeled triglyceride transfer assays were performed as previously described (25 (link), 28 (link), 29 (link), 47 (link)).