Figure 1. MAL expression is required for... | Rockefeller University Press

Figure 1.

$Figure 1. MAL expression is required for translocation of TCR and reorientation of MTOC to IS. (A) Normal Jurkat cells, JTIM cells, and JTIM/MAL cells were extracted with 1% Triton X-100 at 4°C. The soluble (S) and insoluble (I) fractions were isolated by centrifugation to equilibrium in sucrose density gradients. Equivalent aliquots from both fractions were analyzed by immunoblotting with anti-MAL mAb 6D9. The distributions of CD59 and TfR, as respective markers of the insoluble and soluble fractions, were analyzed as a control of the fractionation procedure. (B and C) Cells were conjugated to SEE-pulsed APCs for 15 min. After cell fixation, the distribution of TCR, ICAM-3, and PKC-θ (B) and that of actin, talin, and γ-tubulin (C) were analyzed in nonpermeabilized or permeabilized cells, respectively. The arrows indicate the position of the MTOC in the T cell. (D) The mean percentage ± SEM of Jurkat cells in T cell–APC conjugates with polarized distribution of TCR, actin, and MTOC to the IS was quantified in three independent experiments. n > 100 T cells/experiment (right). Bars, 5 μm.$

MAL expression is required for translocation of TCR and reorientation of MTOC to IS. (A) Normal Jurkat cells, JTIM cells, and JTIM/MAL cells were extracted with 1% Triton X-100 at 4°C. The soluble (S) and insoluble (I) fractions were isolated by centrifugation to equilibrium in sucrose density gradients. Equivalent aliquots from both fractions were analyzed by immunoblotting with anti-MAL mAb 6D9. The distributions of CD59 and TfR, as respective markers of the insoluble and soluble fractions, were analyzed as a control of the fractionation procedure. (B and C) Cells were conjugated to SEE-pulsed APCs for 15 min. After cell fixation, the distribution of TCR, ICAM-3, and PKC-θ (B) and that of actin, talin, and γ-tubulin (C) were analyzed in nonpermeabilized or permeabilized cells, respectively. The arrows indicate the position of the MTOC in the T cell. (D) The mean percentage ± SEM of Jurkat cells in T cell–APC conjugates with polarized distribution of TCR, actin, and MTOC to the IS was quantified in three independent experiments. n > 100 T cells/experiment (right). Bars, 5 μm.