(A) Cav1 and Cav2 protein expression was compared between WT and Cav1-KO CD8 T cell lysates by western blot with anti-Cav1 and Cav2 Ab. Cell lysates were immunoprecipitated with anti-Cav1 and western blots were probed sequentially with anti-Cav1 and Cav2 Ab. (B) Cell lysates were separated into subcellular fractions: C, cytoplasmic; M, membrane soluble; NS, nuclear soluble; CB, chromatin bound; LCC, lipid-cytoskeletal complexes and analysed by Western blot with anti-Cav1 Ab. (C) Total SM content in WT versus Cav1KO CD8 T cells. (D) SM species 14:0 and 16:0 are represented as a percentage of total SM. (E) Free cholesterol from WT and Cav1-KO CD8 T cells is representative of 1 of 3 independent experiments from a total of 12 biological samples of each genotype. (F) Total ceramide and (G) ceramide monohexamide content. Lipidomics data is pooled from 3 biological repeats. Data are shown as mean + SD. NS, not significant; *p ≤0.05, **p ≤0.01, *** p ≤0.001 (Student’s t-test).