Effect of MAL knockdown on IS formation in Jurkat cells and primary T lymphocytes. (A) Jurkat cells were transfected with DNA constructs expressing GFP and a control (c) siRNA or the indicated MAL siRNA and incubated for 48 h at 37°C. GFP-expressing cells were separated in a cell sorter and analyzed for MAL expression by immunoblotting with anti-MAL mAb 6D9. (B) Normal Jurkat cells or Jurkat cells stably expressing either MAL/myc or MALmut/myc were transfected with pMAL-siRNA1/GFP or pMAL-siRNA2/GFP, as indicated. After 48 h at 37°C, cells were conjugated to SEE-loaded Raji cells for 20 min and fixed. The distribution of TCR and MTOC was determined by immunofluorescence analysis. The arrows indicate the position of the MTOC in the T cell. A quantitative analysis of the effect of MAL siRNA expression on MTOC reorientation is shown in the histogram. (C) Jurkat CH7C17 cells expressing GFP and a control siRNA or MAL siRNA1 for 48 h at 37°C were conjugated to either SEB or HA peptide–loaded HOM2 cells for 20 min and fixed. The distribution of TCR was determined by immunofluorescence analysis. (D) Human PBLs were transfected with plasmid DNA expressing GFP and a control siRNA or MAL siRNA1 and incubated for 36 h at 37°C. Cells were then conjugated to SEE-loaded APCs for 15 min. After cell fixation, the distribution of TCR, PKC-θ, and ICAM-3 was determined by immunofluorescence analysis. The histograms show the mean ± SEM of the percentage of APC-conjugated GFP-expressing T cells with MTOC (B) or TCR (C and D) polarized to the IS. Three independent experiments were performed. n > 100 T cells/experiment. Bars, 5 μm.