FIG. 7.

Palmitoylation is not required for LMP2A to block Ca mobilization. BJAB cells were cotransfected with RFP and the indicated LMP2A expression construct or vector control. Ten hours after transfection, the cells were loaded with the calcium-sensitive dye indo-1 1.5 h prior to analysis by flow cytometry. The cells were stimulated with goat anti-human Ig about 40 s after observing baseline calcium levels (indicated by a break in the histogram). Events were collected over 5 min. The x axis represents time, and the y axis represents the ratio of fluorescence at 395 nm to that at 525 nm (ratio of calcium-bound indo-1 to unbound indo-1). Transfected cells were determined by gating on RFP-positive cells. RFP-negative cells represent untransfected cells and serve as an internal control. LMP2B lacks the cytoplasmic amino terminus of LMP2A, and Y74/85F is defective for Syk binding; therefore, both proteins are unable to block BCR signaling. Calcium fluxes of 5-min time courses show representative data of at least three independent experiments.