Figure 1.
Figure 1. INF2 is required for MTOC reorientation in Jurkat cells and primary T cells. (A) Control Jurkat cells (top) and Jurkat cells transiently expressing exogenous, tagged INF2-1 (middle) or INF2-2 (bottom) were conjugated to SEE-loaded APCs. The distribution of α-tubulin, F-actin, and endogenous (top) or exogenous INF2 (middle and bottom) was analyzed. (B–D) Jurkat cells were transfected with DNA constructs coexpressing GFP and shControl, shINF2a, or shINF2b. Cells were processed for immunoblotting with anti-INF2 or anti-GAPDH (GAPDH) antibodies. The histogram represents the percentage of INF2 content (B). Cells were conjugated to SEE-loaded APCs and stained for F-actin and α-tubulin (C). The histogram represents the percentage of T cells with F-actin or MTOC polarized to the IS (D). (E) Peripheral blood lymphocytes transfected with a DNA construct coexpressing GFP and shControl, shINF2a, or shINF2b were conjugated to SEE-loaded APCs and stained for α-tubulin. The histogram represents the percentage of T cells with MTOC polarized to the IS. (F and G) Jurkat cells transiently expressing intact INF2 or the indicated INF2 mutants (F) were transfected with a DNA construct coexpressing GFP and shINF2b. Cells were conjugated to SEE-loaded APCs and the position of the MTOC was analyzed. The histogram represents the percentage of T cells with MTOC polarized to the IS (G). (H) Jurkat cells transiently expressing INF2-1 were transfected with a DNA construct coexpressing GFP and shINF2b. Cells were conjugated to SEE-loaded APCs and stained for INF2-1 and α-tubulin. The arrowheads indicate the position of the MTOC of the T cells. The p-value for the results of DID, DAD, and Cterm1 expression in INF2 KD cells relative to that of INF2 KD cells was of 0.102, 0.086, and 0.138, respectively (G). At least 40 T cells were analyzed in D, E, and G. Quantitative data in B, D, E, and G are summarized as means ± SEM from three independent experiments (error bars; NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars, 5 µm.

INF2 is required for MTOC reorientation in Jurkat cells and primary T cells. (A) Control Jurkat cells (top) and Jurkat cells transiently expressing exogenous, tagged INF2-1 (middle) or INF2-2 (bottom) were conjugated to SEE-loaded APCs. The distribution of α-tubulin, F-actin, and endogenous (top) or exogenous INF2 (middle and bottom) was analyzed. (B–D) Jurkat cells were transfected with DNA constructs coexpressing GFP and shControl, shINF2a, or shINF2b. Cells were processed for immunoblotting with anti-INF2 or anti-GAPDH (GAPDH) antibodies. The histogram represents the percentage of INF2 content (B). Cells were conjugated to SEE-loaded APCs and stained for F-actin and α-tubulin (C). The histogram represents the percentage of T cells with F-actin or MTOC polarized to the IS (D). (E) Peripheral blood lymphocytes transfected with a DNA construct coexpressing GFP and shControl, shINF2a, or shINF2b were conjugated to SEE-loaded APCs and stained for α-tubulin. The histogram represents the percentage of T cells with MTOC polarized to the IS. (F and G) Jurkat cells transiently expressing intact INF2 or the indicated INF2 mutants (F) were transfected with a DNA construct coexpressing GFP and shINF2b. Cells were conjugated to SEE-loaded APCs and the position of the MTOC was analyzed. The histogram represents the percentage of T cells with MTOC polarized to the IS (G). (H) Jurkat cells transiently expressing INF2-1 were transfected with a DNA construct coexpressing GFP and shINF2b. Cells were conjugated to SEE-loaded APCs and stained for INF2-1 and α-tubulin. The arrowheads indicate the position of the MTOC of the T cells. The p-value for the results of DID, DAD, and Cterm1 expression in INF2 KD cells relative to that of INF2 KD cells was of 0.102, 0.086, and 0.138, respectively (G). At least 40 T cells were analyzed in D, E, and G. Quantitative data in B, D, E, and G are summarized as means ± SEM from three independent experiments (error bars; NS, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Bars, 5 µm.

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