MS4a4B is located in lipid rafts. (A-F) T-hybridoma cells expressing the MS4a4B-HA tag were stained with antibodies before FACS analysis as indicated. (A) HA staining of cells untreated (bold line) or treated with Triton X-100 (TX; light line) compared with control vector-infected cells (dashed line). (B) TCR staining of cells untreated (bold line) or TX treated (light line). (C) Thy-1-stained cells untreated (bold line) or TX treated (light line). (D) HA staining for cells untreated (dash line), treated with TX (light line), treated with 10 mM MbCD (bold line), treated with 10 mM MbCD and subsequently TX (shaded line). (E) Thy-1 staining of cells treated as in panel G (MbCD alone not shown). (F) HA staining of cells untreated (dashed line) or treated with sphingomyelinase, 5 U/mL (light line), 10 U/mL (shaded line). (G-H) MS4a4B translocates to lipid rafts in activated spleen cells. Spleen cells were cultured for the indicated times in the presence or absence of concanavalin A (ConA). Cells lysates were separated on discontinuous sucrose gradients, fractionated and selected raft (R) and non-raft (N) fractions were pooled (G), or all fractions were analyzed separately (H). A corresponding amount of the total lysate was reserved before fractionation for comparative purposes (T). Fractions were separated by PAGE and immunoblotted with antibodies to MS4a4B, LAT, or transferrin receptor (CD71) or blotted with labeled cholera toxin B (to detect raft glycoprotein GM1). Results shown are representative of 3 separate experiments.