American Society of Hematology

Figure 2

$Figure 2. INF2 knockdown blocks the exit of MAL vesicles from perinuclear endosomes and decreases the expression of MAL at the plasma membrane. (A-C) Wild-type Jurkat cells (A) or Jurkat cell transiently expressing Cherry-MAL (B-C) were transfected or not (NT) with a DNA construct coexpressing GFP and shControl, or shINF2a or shINF2b, which are targeted to the coding or 3′ untranslated regions, respectively, of INF2 mRNA. Cells were processed for immunoblotting with anti-INF2 antibodies to examine the levels of INF2 or with anti-GADPH antibodies as a loading control (A, top). The 1% Triton-X-100 insoluble (I) membrane fraction of Jurkat cells was separated from the soluble fraction (S), and equivalent aliquots were immunoblotted for MAL and Lck (A bottom). Cells were also processed for fluorescence microscopy to analyze the distribution of MAL (B-C). The intensity of MAL staining in Jurkat cells was measured by densitometric analysis along rectilinear lines as that shown in a representative experiment (C, left). Arrows indicate the position of the periphery of the cell (C middle). The histogram shows the mean percentage ± SEM of cells with low levels of MAL at the plasma membrane as measured as described in “Methods” (C right). Staining intensity was measured in > 50 cells per experiment. Three independent experiments were performed (*P < .05). (D) Jurkat cells transiently expressing Cherry-MAL were transfected with a plasmid coexpressing GFP and shControl or shINF2a for 48 hours, the activity being captured with time-lapse videomicroscopy. Transfected cells were identified by expression of GFP (small top panel). The processes occurring in the cell (large bottom panel) within the boxed region are shown at higher magnification in the right panels. Solid and empty arrowheads indicate 2 vesicles transporting MAL from the pericentriolar region to the plasma membrane or from the plasma membrane to the pericentriolar region, respectively. Numbers indicate time in seconds. Scale bars correspond to 5 μm unless other value is indicated.$

INF2 knockdown blocks the exit of MAL vesicles from perinuclear endosomes and decreases the expression of MAL at the plasma membrane. (A-C) Wild-type Jurkat cells (A) or Jurkat cell transiently expressing Cherry-MAL (B-C) were transfected or not (NT) with a DNA construct coexpressing GFP and shControl, or shINF2a or shINF2b, which are targeted to the coding or 3′ untranslated regions, respectively, of INF2 mRNA. Cells were processed for immunoblotting with anti-INF2 antibodies to examine the levels of INF2 or with anti-GADPH antibodies as a loading control (A, top). The 1% Triton-X-100 insoluble (I) membrane fraction of Jurkat cells was separated from the soluble fraction (S), and equivalent aliquots were immunoblotted for MAL and Lck (A bottom). Cells were also processed for fluorescence microscopy to analyze the distribution of MAL (B-C). The intensity of MAL staining in Jurkat cells was measured by densitometric analysis along rectilinear lines as that shown in a representative experiment (C, left). Arrows indicate the position of the periphery of the cell (C middle). The histogram shows the mean percentage ± SEM of cells with low levels of MAL at the plasma membrane as measured as described in “Methods” (C right). Staining intensity was measured in > 50 cells per experiment. Three independent experiments were performed (*P < .05). (D) Jurkat cells transiently expressing Cherry-MAL were transfected with a plasmid coexpressing GFP and shControl or shINF2a for 48 hours, the activity being captured with time-lapse videomicroscopy. Transfected cells were identified by expression of GFP (small top panel). The processes occurring in the cell (large bottom panel) within the boxed region are shown at higher magnification in the right panels. Solid and empty arrowheads indicate 2 vesicles transporting MAL from the pericentriolar region to the plasma membrane or from the plasma membrane to the pericentriolar region, respectively. Numbers indicate time in seconds. Scale bars correspond to 5 μm unless other value is indicated.

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