American Society of Hematology

Fig. 1.

$Fig. 1. Subcellular distribution of LAMP 1 and 2 and Man 6-P GP in neutrophils. Neutrophils from PB were isolated, disrupted by nitrogen cavitation, and fractionated on a two-layer Percoll Density gradient. (A) LAMP 1 and 2 in subcellular fractions. Other fractions of 1.4 mL were collected and assayed for markers of azurophil granules (MPO, ▪), specific granules (lactoferrin, ▴), gelatinase granules (gelatinase, ▾), secretory vesicles (albumin, X), and plasma membranes (HLA, class I, *). Samples from each fraction were centrifuged to remove Percoll and subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with antibody against LAMP 1 (•) and LAMP 2 (⧫). The immunoblots were developed by chemiluminescence and quantitated by scanning (bold lines). (B) Man 6-P GP in subcellular fractions. Fractions of 1.4 mL were collected and assayed for markers of azurophil granules (MPO, ▪), specific granules (lactoferrin, •), gelatinase granules (gelatinase, ⧫), secretory vesicles (albumin, X ), and plasma membranes (HLA, class I, *). Samples from each fraction were centrifuged to remove Percoll and subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with the sCI-MPR (★) and quantified by phosphorimager (bold line). (C) Man 6-P GP in relation to myeloperoxidase. Isolated neutrophils were disrupted by nitrogen cavitation and the postnuclear supernatant was immunoprecipitated with anti-MPO coupled to sepharose particles, divided into two, and subjected to SDS-PAGE and Coomassie blue staining for protein (left panel) or to SDS-PAGE followed by transfer to nitrocellulose and probed with sCI-MPR (right lane). From left to right: Lane 1, protein profile in immunoprecipitate; lane 2, protein profile in supernatant after immunoprecipitation; lane 3, Man 6-P GP in immunoprecipitate; lane 4, Man 6-P GP in supernatant after immunoprecipitation. (D) Profile of Man 6-P GP in fraction #3 and #10.$

Subcellular distribution of LAMP 1 and 2 and Man 6-P GP in neutrophils. Neutrophils from PB were isolated, disrupted by nitrogen cavitation, and fractionated on a two-layer Percoll Density gradient. (A) LAMP 1 and 2 in subcellular fractions. Other fractions of 1.4 mL were collected and assayed for markers of azurophil granules (MPO, ▪), specific granules (lactoferrin, ▴), gelatinase granules (gelatinase, ▾), secretory vesicles (albumin, X), and plasma membranes (HLA, class I, *). Samples from each fraction were centrifuged to remove Percoll and subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with antibody against LAMP 1 (•) and LAMP 2 (⧫). The immunoblots were developed by chemiluminescence and quantitated by scanning (bold lines). (B) Man 6-P GP in subcellular fractions. Fractions of 1.4 mL were collected and assayed for markers of azurophil granules (MPO, ▪), specific granules (lactoferrin, •), gelatinase granules (gelatinase, ⧫), secretory vesicles (albumin, X ), and plasma membranes (HLA, class I, *). Samples from each fraction were centrifuged to remove Percoll and subjected to SDS-PAGE. The separated proteins were blotted to nitrocellulose membranes and probed with the sCI-MPR (★) and quantified by phosphorimager (bold line). (C) Man 6-P GP in relation to myeloperoxidase. Isolated neutrophils were disrupted by nitrogen cavitation and the postnuclear supernatant was immunoprecipitated with anti-MPO coupled to sepharose particles, divided into two, and subjected to SDS-PAGE and Coomassie blue staining for protein (left panel) or to SDS-PAGE followed by transfer to nitrocellulose and probed with sCI-MPR (right lane). From left to right: Lane 1, protein profile in immunoprecipitate; lane 2, protein profile in supernatant after immunoprecipitation; lane 3, Man 6-P GP in immunoprecipitate; lane 4, Man 6-P GP in supernatant after immunoprecipitation. (D) Profile of Man 6-P GP in fraction #3 and #10.

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