Figure 6 LAMP1 is required for efficient... | American Society of Hematology

Figure 6

$Figure 6. LAMP1 is required for efficient delivery of perforin, but not granzyme B, to lytic granules. (A) YTS cells, transduced with CTRL or LAMP1 RNAi, were homogenized and fractionated on a continuous 0% to 30% gradient of iodoxanol. The presence of the indicated proteins in the gradient fractions was assessed by immunoblotting; only relevant fractions are shown. The data are representative of 3 experiments. EEA-1 was used as a marker of early endosomes; Rab9, late endosomes; Rab7, late endosomes/lysosomes; and LAMP2, lysosomes. Adaptin γ and MPR were used as markers of TGN-derived transport vesicles. (B) YTS cells were fixed, stained with anti–CI-MPR Ab followed by DyLight 649–conjugated anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin D48 Ab (green). The area of colocalization between the 2 fluorophores is shown as a heat map image. Scale bars represent 5 μm. Inserts show DIC images. (C) The percentage of colocalization between perforin (D48-reactive) and CI-MPR. The data were determined by analysis of 34 to 35 cells, as described in (B), and are shown as mean values + SD from 2 experiments. The numbers below the graph are Manders overlap coefficients ± SD. ***P < .001; Mann-Whitney U test.$

LAMP1 is required for efficient delivery of perforin, but not granzyme B, to lytic granules. (A) YTS cells, transduced with CTRL or LAMP1 RNAi, were homogenized and fractionated on a continuous 0% to 30% gradient of iodoxanol. The presence of the indicated proteins in the gradient fractions was assessed by immunoblotting; only relevant fractions are shown. The data are representative of 3 experiments. EEA-1 was used as a marker of early endosomes; Rab9, late endosomes; Rab7, late endosomes/lysosomes; and LAMP2, lysosomes. Adaptin γ and MPR were used as markers of TGN-derived transport vesicles. (B) YTS cells were fixed, stained with anti–CI-MPR Ab followed by DyLight 649–conjugated anti-mouse Ab (red), and then stained with Alexa Fluor 488–conjugated anti-perforin D48 Ab (green). The area of colocalization between the 2 fluorophores is shown as a heat map image. Scale bars represent 5 μm. Inserts show DIC images. (C) The percentage of colocalization between perforin (D48-reactive) and CI-MPR. The data were determined by analysis of 34 to 35 cells, as described in (B), and are shown as mean values + SD from 2 experiments. The numbers below the graph are Manders overlap coefficients ± SD. ***P < .001; Mann-Whitney U test.

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