FIG. 2.
Palmitylation of human CAR at cysteines 259 and 260. (A) Control CHO cells (lanes 1 and 2) and CHO-CAR cells (lanes 3 and 4) were starved for 1 h at 37°C in Dulbecco modified Eagle medium containing 2% dialyzed fetal bovine serum and radiolabeled for 4 h at 37°C with 125I-IC16, an iodinated palmitate analog, at 20 μCi/ml (top). In parallel, cells were incubated for 1 h at 37°C in Dulbecco modified Eagle medium minus methionine and cysteine containing 2% dialyzed fetal bovine serum and radiolabeled for 4 h at 37°C with Expre35S35S protein labeling mix at 25 μCi/ml (35S-Met; bottom). Lysates from duplicate radiolabeled dishes were subjected to immunoprecipitation with anti-CAR monoclonal antibody (RmcB) and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Radiolabeled CAR migrated with an apparent molecular mass of 46 kDa, as indicated by the arrow. (B) COS-1 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, Calif.) and empty pcDNA3.1 vector (Mock; lanes 1 and 2) or plasmids containing full-length CAR (lanes 3 and 4), C259A, C260A- CAR (lanes 5 and 6), tailless-CAR (lanes 7 and 8), or C259Stop-CAR (lanes 9 and 10) and radiolabeled with 125I-IC16 (top) and Expre35S35S (35S-Met; bottom). The tailless-CAR and C259Stop-CAR constructs migrated at 33 kDa, as indicated by the arrow.