American Society of Hematology

Figure 1

$Figure 1. The 72-5ptase is expressed in RAW264.7 macrophages and inhibits FcγR-mediated phagocytosis. (A) (i) 72-5ptase domain structure, showing proline-rich (PxxP) motifs (■), catalytic domain (▒), D480N mutation which renders the enzyme inactive (↓) and peptide used to raise polyclonal antibodies. (ii) Cytosol, Triton-X-100-soluble (Tx100 sol) and -insoluble (Tx100 insol) membrane fractions from RAW264.7 macrophages were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with 72-5ptase-specific antibodies. (iii) RAW264.7 Triton-X-100-soluble fraction was immunoprecipitated (IP) with nonimmune or immune 72-5ptase-specific antibodies and immunoblotted (IB) with anti-72-5ptase antibodies. MW, molecular weight. RAW264.7 macrophages ectopically expressing (B and D) vector, HA-72-5ptase, HA-SHIP1, or HA-72-D480N5ptase, or (C) vector, HA-72-5ptase, or HA-72-D480N5ptase were stimulated to phagocytose (B) eIgG, (C) 3-bIgG and 10-bIgG (−/+ wortmannin), or (D) SOZ. The phagocytic index represents the number of particles phagocytosed per 100 cells for at least 3 independent experiments, normalized to vector-expressing cells, designated 100%. Data are expressed as the mean (± SEM) of at least 3 independent experiments (*P < .05). (E) RAW264.7 macrophages were cotransfected with GFP-VAMP3 and vector, HA-72-5ptase, or HA-72-D480N5ptase, and phagocytosis was stimulated by the addition of eIgG. (i) Images represent a time course of phagocytosis in cells expressing vector, HA-72-5ptase, or HA-72-D480N5ptase imaged by confocal microscopy. Expression of GFP-VAMP3 is shown. Insets show enlargements of boxed areas. Closed arrowheads indicate forming phagocytic cups, and open arrowheads indicate GFP-VAMP3-positive vesicle recruitment. Scale bar represents 20 μm. In the middle panels closed arrowheads indicate distorted phagocytic cup in HA-72-5ptase-expressing cell. Time of phagocytic cup closure was designated 0 seconds. (ii) Time for phagosome closure was determined by scoring 16 to 20 phagocytic events over 4 independent experiments for each construct. Data represent mean (± SEM) time of phagosome closure from the commencement of phagocytic cup formation (sec) of 4 independent experiments. *P < .05.$

The 72-5ptase is expressed in RAW264.7 macrophages and inhibits FcγR-mediated phagocytosis. (A) (i) 72-5ptase domain structure, showing proline-rich (PxxP) motifs (■), catalytic domain (▒), D480N mutation which renders the enzyme inactive (↓) and peptide used to raise polyclonal antibodies. (ii) Cytosol, Triton-X-100-soluble (Tx100 sol) and -insoluble (Tx100 insol) membrane fractions from RAW264.7 macrophages were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with 72-5ptase-specific antibodies. (iii) RAW264.7 Triton-X-100-soluble fraction was immunoprecipitated (IP) with nonimmune or immune 72-5ptase-specific antibodies and immunoblotted (IB) with anti-72-5ptase antibodies. MW, molecular weight. RAW264.7 macrophages ectopically expressing (B and D) vector, HA-72-5ptase, HA-SHIP1, or HA-72-D480N5ptase, or (C) vector, HA-72-5ptase, or HA-72-D480N5ptase were stimulated to phagocytose (B) eIgG, (C) 3-bIgG and 10-bIgG (−/+ wortmannin), or (D) SOZ. The phagocytic index represents the number of particles phagocytosed per 100 cells for at least 3 independent experiments, normalized to vector-expressing cells, designated 100%. Data are expressed as the mean (± SEM) of at least 3 independent experiments (*P < .05). (E) RAW264.7 macrophages were cotransfected with GFP-VAMP3 and vector, HA-72-5ptase, or HA-72-D480N5ptase, and phagocytosis was stimulated by the addition of eIgG. (i) Images represent a time course of phagocytosis in cells expressing vector, HA-72-5ptase, or HA-72-D480N5ptase imaged by confocal microscopy. Expression of GFP-VAMP3 is shown. Insets show enlargements of boxed areas. Closed arrowheads indicate forming phagocytic cups, and open arrowheads indicate GFP-VAMP3-positive vesicle recruitment. Scale bar represents 20 μm. In the middle panels closed arrowheads indicate distorted phagocytic cup in HA-72-5ptase-expressing cell. Time of phagocytic cup closure was designated 0 seconds. (ii) Time for phagosome closure was determined by scoring 16 to 20 phagocytic events over 4 independent experiments for each construct. Data represent mean (± SEM) time of phagosome closure from the commencement of phagocytic cup formation (sec) of 4 independent experiments. *P < .05.

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