American Society of Hematology

Figure 5.

$Figure 5. Intracellular mistrafficking and cytoplasmic accumulation of MT-HNE proteins in neutrophil granulocytes of patients with SCN. (A) Localization of HNE protein (29-34 kDa) in subcellular fractions of primary polymorphonuclear cell lysates obtained by differential centrifugation. Analysis of the following fractions: P1 (nuclear and nuclear-associated proteins), P2 (mitochondria, lysosomes, ER), P3 (plasma membrane, secretory and other vesicles, Golgi complex), and S (soluble cytosolic proteins). TL indicates total cell lysate. A healthy donor without G-CSF treatment and a healthy donor who received G-CSF subcutaneously for 2 days served as controls. Both SCN patients received daily treatment with G-CSF. Each patient carries a heterozygous mutation in ELA2 (patient 1, C122Y; patient 2, ΔV161-F170). Localization of cellular marker proteins in subcellular fractions was obtained by differential centrifugation. Nucleoporin (62 kDa) is a nuclear and cytoplasmic pore complex protein, thus defining P1, P3, and S. Lamp-2 (120 kDa) is a lysosomal membrane protein that fractionates mainly in P2, and CD45 (180-220 kDa) is a plasma membrane–associated protein mainly localized in fraction P3. (B) Subcellular localization of WT-HNE in granulocytes of healthy donors with or without rh-G-CSF treatment or of MT-HNE in granulocytes of a patient with SCN and carrying a heterozygous mutation in ELA2 (patient 2, ΔV161-F170). Cells were stained with anti-HNE (green) and lamp-3 (lysosomal marker; red). TO-PRO-3 was used to counterstain the nuclei (blue). Yellow represents colocalization of HNE protein with lamp-3. (C) Subcellular localization of MPO in granulocytes of healthy donors with or without rh-G-CSF treatment or in granulocytes of a patient with SCN and carrying a heterozygous mutation in ELA2 (patient 2, ΔV161-F170). Cells were stained with anti-MPO (red). TO-PRO-3 was used to counterstain the nuclei (blue).$

Intracellular mistrafficking and cytoplasmic accumulation of MT-HNE proteins in neutrophil granulocytes of patients with SCN. (A) Localization of HNE protein (29-34 kDa) in subcellular fractions of primary polymorphonuclear cell lysates obtained by differential centrifugation. Analysis of the following fractions: P1 (nuclear and nuclear-associated proteins), P2 (mitochondria, lysosomes, ER), P3 (plasma membrane, secretory and other vesicles, Golgi complex), and S (soluble cytosolic proteins). TL indicates total cell lysate. A healthy donor without G-CSF treatment and a healthy donor who received G-CSF subcutaneously for 2 days served as controls. Both SCN patients received daily treatment with G-CSF. Each patient carries a heterozygous mutation in ELA2 (patient 1, C122Y; patient 2, ΔV161-F170). Localization of cellular marker proteins in subcellular fractions was obtained by differential centrifugation. Nucleoporin (62 kDa) is a nuclear and cytoplasmic pore complex protein, thus defining P1, P3, and S. Lamp-2 (120 kDa) is a lysosomal membrane protein that fractionates mainly in P2, and CD45 (180-220 kDa) is a plasma membrane–associated protein mainly localized in fraction P3. (B) Subcellular localization of WT-HNE in granulocytes of healthy donors with or without rh-G-CSF treatment or of MT-HNE in granulocytes of a patient with SCN and carrying a heterozygous mutation in ELA2 (patient 2, ΔV161-F170). Cells were stained with anti-HNE (green) and lamp-3 (lysosomal marker; red). TO-PRO-3 was used to counterstain the nuclei (blue). Yellow represents colocalization of HNE protein with lamp-3. (C) Subcellular localization of MPO in granulocytes of healthy donors with or without rh-G-CSF treatment or in granulocytes of a patient with SCN and carrying a heterozygous mutation in ELA2 (patient 2, ΔV161-F170). Cells were stained with anti-MPO (red). TO-PRO-3 was used to counterstain the nuclei (blue).

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