CD44 palmitoylation-impaired constructs have reduced affiliation with lipid rafts. (A) Schematic representation of palmitoylation-impaired CD44 constructs generated by site-directed mutagenesis of human CD44. Single-site mutants were termed C286A and C286S, while double-site mutants (C286S + C295A, C286A + C295A) were termed SA and AA respectively. (B) MDA-MB-231 cells were transfected with full-length CD44 (CD44WT) or CD44 single-site (C286A, C286S) and double-site (SA, AA) palmitoylation-impaired mutants and were selected for 48 hours. Whole cell lysates revealed increased expression of CD44 in the transfected cells. (C) Full-scale lipid raft extractions confirmed reductions in raft-affiliated CD44 in cells expressing the mutant constructs relative to controls. (D) Calculation of the lipid raft:nonlipid raft (LR:NR) ratio for multiple experiments confirmed statistically significant reductions in CD44 raft affiliation upon expression of the CD44 palmitoylation-impaired mutants. (E) Triton X-100-insoluble and Triton X-100-soluble fractions were isolated and confirmed to be enriched in respectively lipid raft (Flotillin-1 (Flot-1)) or nonraft (transferrin receptor (TfR)) markers. CD44 recovery from raft-enriched fractions was reduced in cells overexpressing mutant CD44, and paralleled by increased recovery of CD44 in nonraft fractions. (F) Calculation of the affiliation ratio of CD44 with detergent-insoluble versus detergent-soluble fractions confirmed reductions in raft-affiliated CD44 from cells expressing mutant constructs relative to controls. Error bars, standard error of the mean; n = 3.#P < 0.05 (C286S vs. control); *P < 0.05; **P < 0.01, Student’s t test. HAM, hydroxylamine; WT, wild-type.