Anti cers2

DMD patient myoblast cells or mouse skeletal muscle tissues were lysed on ice in radioimmunoprecipitation assay buffer composed of 50 mM tris-HCl, 5 M NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 100 mM NaF, sodium deoxycholate (5 mg/ml), and 1% NP-40 containing protease and phosphatase inhibitors (Roche). Protein concentrations were determined using the Bradford method, and samples were loaded on a 12% SDS–polyacrylamide gel electrophoresis (PAGE) gel. After electrophoresis, proteins were separated by SDS-PAGE and transferred onto methanol-activated polyvinylidene difluoride membranes. Blocking of the membranes was done in 5% milk-TBST (tris-buffered saline with 0.1% Tween 20) for 1 hour, and after washing, the membranes were incubated overnight with primary antibody anti-SPTLC1 (Proteintech) or anti-SPTLC2 (Thermo Fisher Scientific) or anti-CERS2 (Sigma-Aldrich) in 3% BSA-TBST (1:1000). Incubation with secondary anti-rabbit polyclonal antibody was done in 5% BSA-TBST (1:2000). Antibody detection reactions were developed by enhanced chemiluminescence (Advansta) and imaged using the c300 Imaging System (Azure Biosystems).

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.