American Society of Hematology

Fig. 3.

$Fig. 3. p47phox is enriched on nascent and mature PMN phagosomes. (A) Adherent PMNs ingested OpZ for 0.5 or 15 minutes at 37°C before processing for IFM. Fixed cells were double-stained with MoAbs to p57 and pAbs to p47phox followed by secondary antibodies coupled to FITC and Texas red, respectively. p47phoxand p57 colocalized on forming phagosomes (upper panels) and p47phox was retained after p57 was shed (lower panels). Arrowhead, uningested OpZ; arrows, phagosomes; Asterisk, representative nuclear lobe that was not stained with anti-p57 MoAbs. (B) Kinetics of p47phox association with OpZ phagosomes. Adherent PMNs ingested OpZ for 0 to 15 minutes at 37°C. Fixed cells were double-stained with pAbs to p47phox and MoAbs to p57 followed by secondary antibodies conjugated to FITC or Texas red, respectively. Data shown are the mean ± SD from four independent controls assayed in duplicate or triplicate. At least 100 phagosomes were scored/sample/time. (•), p57; (○), p47phox. (C) F-actin, p57, and p47phox are enriched on purified OpZ phagosomes. OpZ phagosomes were purified on sucrose gradients and proteins in the phagosome fractions were detected by SDS-PAGE and immunoblotting. Note that OpZ phagosomes (lanes 3 and 4) were enriched for p57, F-actin, and p47phox. By contrast, these proteins did not bind to unopsonized zymosan (Z) particles that were not ingested. Data shown are representative of three independent experiments.$

p47phox is enriched on nascent and mature PMN phagosomes. (A) Adherent PMNs ingested OpZ for 0.5 or 15 minutes at 37°C before processing for IFM. Fixed cells were double-stained with MoAbs to p57 and pAbs to p47phox followed by secondary antibodies coupled to FITC and Texas red, respectively. p47phoxand p57 colocalized on forming phagosomes (upper panels) and p47phox was retained after p57 was shed (lower panels). Arrowhead, uningested OpZ; arrows, phagosomes; Asterisk, representative nuclear lobe that was not stained with anti-p57 MoAbs. (B) Kinetics of p47phox association with OpZ phagosomes. Adherent PMNs ingested OpZ for 0 to 15 minutes at 37°C. Fixed cells were double-stained with pAbs to p47phox and MoAbs to p57 followed by secondary antibodies conjugated to FITC or Texas red, respectively. Data shown are the mean ± SD from four independent controls assayed in duplicate or triplicate. At least 100 phagosomes were scored/sample/time. (•), p57; (○), p47phox. (C) F-actin, p57, and p47phox are enriched on purified OpZ phagosomes. OpZ phagosomes were purified on sucrose gradients and proteins in the phagosome fractions were detected by SDS-PAGE and immunoblotting. Note that OpZ phagosomes (lanes 3 and 4) were enriched for p57, F-actin, and p47phox. By contrast, these proteins did not bind to unopsonized zymosan (Z) particles that were not ingested. Data shown are representative of three independent experiments.

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