Fig. 2.

SCIMP is a palmitoylated transmembrane protein present in Brij 98- but not NP-40-resistant membrane microdomains. (A) Fractionation of Ramos cells into cytoplasmic (Cyt.), nuclear (Nuc.), and membrane (Mem.) fractions, followed by SCIMP immunoblotting. NTAL and Erk2 staining served as controls for membrane and cytoplasmic fractions, respectively. (B) Acyl-biotinyl exchange reaction performed on lysates (Lys.) from membrane fractions of Ramos cells. Non-hydroxylamine-treated samples (−HA) served as a negative control, while HA treatment (+HA) allowed exchange of palmitate for biotin moiety and isolation of palmitoylated proteins on immobilized streptavidin. Lyn and Igμ staining, respectively, served as positive and negative controls for specificity of isolation. (C and D) Brij 98 or NP-40 lysates from Ramos cells were fractionated by flotation in sucrose density gradient (C) or by gel filtration on Sepharose 4B (D). Samples were analyzed for distribution of SCIMP, CD37, MHC-II, and PAG. Data are representative of two independent experiments.