Figure 4.

EWI-2 affects cell surface molecular organization of CD9 and CD81. (A) Intact U87-LucNeo cells (expressing wild type EWI-2 or vector control) were labeled with biotin and were then lysed. CD9, CD81, and EWI-2 were immunoprecipitated, and cell surface proteins were visualized by blot analysis with ExtrAvidin. Note: EWI-2-associated CD9 and CD81 themselves do not label very well with biotin because associated proteins limit accessibility (e.g., see Kolesnikova et al. [3]). (B) U87-LucNeo cells, expressing vector alone or EWI-2, were incubated with mAb W6/32 (to MHC-I), mAb C9BB (which preferentially recognizes oligomerized CD9 [18], or mAb MM2/57 (which recognizes total CD9). After washing, bound antibodies were visualized using fluorescein isothiocyanate-rabbit antimouse second antibody, and quantitated by flow cytometry. R = ratio of mean fluorescence intensities for C9BB divided by MM2/57. (C) U87-LucNeo cells, expressing vector alone or EWI-2, were labeled with ³H-palmitate, lysed, and then CD9, CD81, and EWI-2 were immunoprecipitated. Lower panels show an enlarged view of key regions from the top panel. The palmitoylation of EWI-2 on membrane proximal cysteines will be described in more detail elsewhere. (D) Densitometric scans of lanes 1 to 4 are shown. Quantitation reveals increases of approximately three-fold in recovery of CD9 (in lane 4 vs 3) and CD81 (in lane 2 vs 1). Note: These results provide the first definitive demonstration that EWI-2 is palmitoylated.