American Society of Hematology

Fig. 5.

$Fig. 5. Expression of NRAMP1 in human neutrophils granules after PMA treatment. / Prior to disruption, cells were either kept on ice (control; lane −) or stimulated for 15 minutes at 37°C with 2μg/mL PMA (lane +). After washing and disruption of the cells, the postnuclear supernatants were centrifuged on a 3-layer Percoll gradients, fractionated, and pooled in 4 samples 1, 2, 3, and 4, as described in Figure 3. Pooled fractions 1, 2, 3, and 4 were assayed by ELISA for myeloperoxidase, lactoferrin, albumin (not shown), and gelatinase (A) and identified as α-, β1-, β2-, and γ-bands. The same fractions (25 μg) were separated by SDS-PAGE on a 10% acrylamide gel followed by transfer to PVDF membrane (B,C). Immunoblotting was performed with antibodies raised against gelatinase (B) and NRAMP1 (C). The positions and sizes (in kilodaltons) of the molecular weight markers are shown.$

Expression of NRAMP1 in human neutrophils granules after PMA treatment.

Prior to disruption, cells were either kept on ice (control; lane −) or stimulated for 15 minutes at 37°C with 2μg/mL PMA (lane +). After washing and disruption of the cells, the postnuclear supernatants were centrifuged on a 3-layer Percoll gradients, fractionated, and pooled in 4 samples 1, 2, 3, and 4, as described in Figure 3. Pooled fractions 1, 2, 3, and 4 were assayed by ELISA for myeloperoxidase, lactoferrin, albumin (not shown), and gelatinase (A) and identified as α-, β1-, β2-, and γ-bands. The same fractions (25 μg) were separated by SDS-PAGE on a 10% acrylamide gel followed by transfer to PVDF membrane (B,C). Immunoblotting was performed with antibodies raised against gelatinase (B) and NRAMP1 (C). The positions and sizes (in kilodaltons) of the molecular weight markers are shown.

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