Figure 3. MAL is required for the... | Rockefeller University Press

Figure 3.

$Figure 3. MAL is required for the targeting of Lck to the plasma membrane. (A) The subcellular distribution of endogenous Lck was analyzed by indirect immunofluorescence in the three types of Jurkat cell. The percentage of cells with low levels of peripheral Lck are shown on the right and measured as described in Materials and methods. (B) Human PBLs were transfected with plasmid DNA expressing GFP and MAL siRNA1 and incubated for 36 h at 37°C. After cell fixation, the distribution of Lck was determined by immunofluorescence analysis in three independent experiments. n > 100 T cells/experiment. The histograms represent the mean percentage ± SEM of cells with predominant intracellular localization of Lck. (C) The soluble (S) and insoluble (I) membrane fractions of resting normal Jurkat cells, JTIM cells, and JTIM/MAL cells were isolated by centrifugation to equilibrium. Equivalent aliquots from both fractions were then analyzed by immunoblotting to detect endogenous Lck and LAT. The histogram represents the mean percentage ± SEM of Lck into the membrane insoluble fraction. Three independent experiments were performed. (D) Cells were conjugated to SEE-pulsed APCs for 15 min. After cell fixation, the distribution of Lck was analyzed. The histogram represents the mean percentage of Jurkat cells in T cell–APC conjugates with polarized distribution of Lck to the IS as quantified in three independent experiments. n > 100 T cells/experiment. (E) Lysates of normal Jurkat cells or Jurkat cells stably expressing MAL/myc cells were immunoprecipitated with anti-myc mAb, and the immunoprecipitates and the original lysates were analyzed by immunoblotting with antibodies to the c-myc tag or to Lck as indicated (left). Normal COS-7 cells or COS-7 cells transiently coexpressing MAL/myc and Lck-GFP, Lck10-GFP, p75-GFP, or GFP were lysed and immunoprecipitated with anti-myc or GFP antibodies. The immunoprecipitates and the original lysates were finally analyzed by immunoblot with anti-myc and anti-GFP antibodies as indicated (right). Bars, 5 μm.$

MAL is required for the targeting of Lck to the plasma membrane. (A) The subcellular distribution of endogenous Lck was analyzed by indirect immunofluorescence in the three types of Jurkat cell. The percentage of cells with low levels of peripheral Lck are shown on the right and measured as described in Materials and methods. (B) Human PBLs were transfected with plasmid DNA expressing GFP and MAL siRNA1 and incubated for 36 h at 37°C. After cell fixation, the distribution of Lck was determined by immunofluorescence analysis in three independent experiments. n > 100 T cells/experiment. The histograms represent the mean percentage ± SEM of cells with predominant intracellular localization of Lck. (C) The soluble (S) and insoluble (I) membrane fractions of resting normal Jurkat cells, JTIM cells, and JTIM/MAL cells were isolated by centrifugation to equilibrium. Equivalent aliquots from both fractions were then analyzed by immunoblotting to detect endogenous Lck and LAT. The histogram represents the mean percentage ± SEM of Lck into the membrane insoluble fraction. Three independent experiments were performed. (D) Cells were conjugated to SEE-pulsed APCs for 15 min. After cell fixation, the distribution of Lck was analyzed. The histogram represents the mean percentage of Jurkat cells in T cell–APC conjugates with polarized distribution of Lck to the IS as quantified in three independent experiments. n > 100 T cells/experiment. (E) Lysates of normal Jurkat cells or Jurkat cells stably expressing MAL/myc cells were immunoprecipitated with anti-myc mAb, and the immunoprecipitates and the original lysates were analyzed by immunoblotting with antibodies to the c-myc tag or to Lck as indicated (left). Normal COS-7 cells or COS-7 cells transiently coexpressing MAL/myc and Lck-GFP, Lck₁₀-GFP, p75-GFP, or GFP were lysed and immunoprecipitated with anti-myc or GFP antibodies. The immunoprecipitates and the original lysates were finally analyzed by immunoblot with anti-myc and anti-GFP antibodies as indicated (right). Bars, 5 μm.