Ecl detection system

NBD-Sphinganine and fatty acyl CoAs were from Avanti Polar Lipids. Defatted-bovine serum albumin, a protease inhibitor cocktail, anti-HA, anti-CerS2, and anti-tubulin antibodies were from Sigma-Aldrich. Protein A agarose beads and anti-CerS6 antibodies were from Santa Cruz. Horseradish peroxidase was from the Jackson Laboratory. An ECL detection system was from Cyanagen. Silica gel 60 thin layer chromatography plates were from Merck. All solvents were of analytical grade and purchased from Bio-Lab.

NBD-Sphinganine (NBD-Sph) and fatty acyl-CoAs were from Avanti Polar Lipids (Alabaster, AL). Defatted BSA, a protease inhibitor mixture, and polyethyleneimine were from Sigma. An ECL detection system and a BCA reagent kit were from Cyanagen (Bologna, Italy). Silica gel 60 TLC plates were from Merck (Billerica, MA). All solvents were of analytical grade and were purchased from Bio-Lab (Jerusalem, Israel).

Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Hemagglutinin-tagged CerS2 was immunoprecipitated using a rabbit anti-HA antibody and protein A agarose beads. CerS6 was identified using a mouse anti-CerS6 antibody (1:1000; Santa Cruz) and goat anti-mouse horseradish peroxidase (1:10,000; Jackson Laboratory) as the secondary antibody. CerS2 was detected using a rabbit anti-CerS2 antibody (1:5000; Sigma) followed by incubating with goat anti-rabbit horseradish peroxidase (1:10,000) as the secondary antibody. Equal loading was confirmed using a mouse anti-tubulin antibody (1:5000; Sigma). Detection was performed using an ECL detection system (Cyanagen).