INF2 is required for Glu-MT formation in T cells. (A and B) Jurkat cells were transfected with DNA constructs coexpressing GFP and shControl, shINF2a, or shINF2b. Cells were conjugated to SEE-loaded APCs and stained for Glu- and Tyr-MTs or acetyl- and Tyr-MTs, as indicated (A). The histogram represents the percentage of T cells with Glu- or acetyl-MTs (B). (C) Primary T cells were transfected with DNA constructs coexpressing GFP and shControl, shINF2a, or shINF2b. Cells were then conjugated to SEE-loaded APCs and stained for Glu- and Tyr-MTs. The histogram represents the percentage of T cells with Glu-MTs. (D) Jurkat cells expressing shControl, shINF2a, shINF2b, or shINF2b and exogenous INF2-1 were conjugated to SEE-loaded APCs and immunoblotted for Glu- or total α-tubulin. The histogram represents the percentage of Glu-tubulin content. (E) Jurkat cells expressing GFP and shINF2b from the same plasmid were either cotransfected with DNA constructs expressing the intact FH2 domain of INF2 or the I/K mutant, or were treated with 3 nM taxol for 18 h. Cells were then conjugated with SEE-loaded APCs, fixed with methanol, and stained for the expressed INF2 FH2 fragment and for Glu- or total α-tubulin, as indicated. The histogram represents the percentage of T cells with Glu-MTs or with polarized MTOC. The arrowheads in C and E indicate the position of the MTOC of the T cells. At least 40 T cells were analyzed in B, C, and E. Quantitative data in B–E are summarized as means ± SEM from three independent experiments (error bars; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars, 5 µm.