Decreased CD44 palmitoylation is paralleled by increased co-association with ezrin. (A) CD44 immunoprecipitates from different MDA-MB-231 migration time points were probed for palmitoylated CD44 by 1-biotinamido-4-(4′-(maleimidomethyl)cyclohexanecarboxamido)butane (BMCC) assay. Palmitoylated CD44 was detected using streptavidin (CD44-Palm), and total CD44 detected using a CD44 primary antibody (CD44-Total). The absence of hydroxylamine (HAM) treatment was a negative control for palmitoylated CD44. CD44-Palm levels decreased over the migration time course. (B) Quantification of CD44-Palm as a ratio of CD44-Total revealed significant time-dependent reductions during migration. Error bars, standard error of the mean; n = 3 experiments. *P < 0.05, Student’s t test. (C) CD44 was immunoprecipitated from whole cell lysates of MDA-MB-231 cells at the indicated migration time points, with IgG as an internal control. Immunoprecipitates were probed for CD44 and ezrin, with the IgG heavy chain band intensity considered as a loading control. Ezrin-CD44 co-immunoprecipitation (IP) was increased after 1 and 2 hours of cell migration compared with nonmigrating cells (time = 0). (D) CD44 was immunoprecipitated from migrating (2 hours) untransfected MDA-MB-231 (Ctrl), WT-expressing, single and double palmitoylation mutant-expressing cells. The latter cells had a notable increase in CD44-ezrin co-association. IP experiments are representative of three independent experiments. (E) Confluent breast primary cells were stained for CD44 (green) and Flotillin-1 (Flot-1, red). Nuclei were stained with DAPI and are shown in grey. Ductal carcinoma in situ (DCIS) primary cells demonstrated the highest co-localisation of the two proteins compared with invasive ductal carcinomas (IDC). (F) Whole cell lysates of primary cultures from two nontumour (NT), and six IDC of tumour grade 2 and 3 (three of each) were subjected to BMCC assays to compare palmitoylated CD44 levels. Most palmitoylated CD44 (CD44-Palm) was recovered from NT cultures, while all patient IDC cultures displayed comparatively little CD44-Palm. Palmitoylated CD44 levels densitometrically normalised to total CD44 (CD44-Total) further demonstrated increased levels of CD44-Palm in NT samples. Error bars, standard deviation of duplicates in one experiment.