Ecl select western blot detection system

Protein samples were resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (GE Healthcare UK Ltd., Buckinghamshire, England) using a Trans-Blot SD semi-dry transfer cell (Bio-Rad). Membranes were stained with Ponceau S (Sigma–Aldrich Co. LLC., St. Louis, MO), and then blocked overnight with 5% skim milk (BD Biosciences, Franklin Lakes, NJ) in Tris-buffered saline with 0.1% Tween 20 (Wako) (TBST). Primary antibodies were diluted in 5% skim milk/TBST as following: anti-LPCAT3 (40 ng/ml), anti-FLAG M2 antibody (5 μg/ml, Sigma–Aldrich), anti-MTP antibody (50 ng/ml, BD), or anti-PDI antibody (1:1000 dilution, Cell Signaling Technology, Inc., Danvers, MA). Horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) were used at a 1:2000 dilution in 5% skim milk/TBST. TBST was used for washing steps and changed at least three times between incubation steps. ECL select western blot detection system (GE Healthcare) was used for chemiluminescence, and detected using ImageQuant LAS500 (GE Healthcare).

Protein samples were resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes (GE Healthcare, Tokyo, Japan) using a Trans-Blot transfer cell (Bio-Rad). Membranes were blocked for more than 16 h with 5% skim milk (BD Biosciences, Franklin Lakes, NJ, USA). Anti-LPCAT2 (1:1000), anti-phospho-LPCAT2 (1:1000), and anti-calnexin (1:100) (BD Biosciences) antibodies were used as the primary antibodies. Anti-LPCAT2 and anti-phospho-LPCAT2 antibodies were available from another study (13 (link)). Horseradish peroxidase–conjugated secondary antibodies (1:2000; GE Healthcare) were used. ECL select Western blot detection system (GE Healthcare) was used for chemiluminescence, and detected using ImageQuant LAS500 (GE Healthcare).