Figure 1.

Endogenously expressed NTSR-1 in breast cancer cells is palmitoylated. (A) MDA-MB-231 cells were seeded in 10 cm plates followed by NTSR-1 immunoprecipitation. After receptor immunoprecipitation, free cysteine residues were protected using Nethylmaleimide (NEM) followed by NH₂OH-mediated cleavage of palmitate groups. The free cysteine residues were then labeled with biotin using biotin-conjugated 1-biotin-amido-4-[4-(maleimidomethyl)cyclohexanecarboxamido]butane (BMCC) and immunoprecipitates were analyzed using SDS electrophoresis using an avidin antibody to detect the biotinylated labeled palmitoylated residues. Representative blots from three independent experiments. (B) MDA-MB-231 cells were seeded in 10 cm plates. After 24 h, cells were treated with 10 µM 2-BP followed by analysis using fatty acyl exchange labeling assay as described above. Representative blots from two independent experiments. As described previously in reference ¹⁹, under these immunoprecipitation conditions, the high molecular weight NTSR-1 isoform (∼54 kDa) is preferentially precipitated.