American Society of Hematology

Figure 5

$Figure 5. BLT1 phosphorylation by Src is required for IgG-induced FcγRI-BLT1 association in LRs. (A) AMs were incubated with or without 30:1 IgG-RBCs for 10 minutes and subjected to membrane fractionation. LR and non-LR membrane fractions were pooled and immunoprecipitated with an antibody against BLT1, after which immunoprecipitates were subjected to immunoblot analysis for the proteins shown on the right. (B) Relative distribution of total BLT1 as well as tyrosine- and serine-phosphorylated BLT1 was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel A, and is expressed as the LR/non-LR ratio of each. *P < .05 vs control. (C) AMs were pretreated with or without the Src kinase inhibitor PP2 (1 μM) or its negative control congener PP3 (1 μM) for 20 minutes, followed by incubation with or without 30:1 IgG-RBCs for 10 minutes. Pooled LR and non-LR fractions were prepared and subjected to immunoblot analysis for the proteins indicated at the right. (D) Relative distribution of BLT1 and FcγRI was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel C, and is expressed as the LR/non-LR ratio of each. *P < .05 vs control, and #P < .05 vs IgG-RBC. (E) AMs were incubated with or without PP2 or PP3 for 20 minutes, followed by challenge with 30:1 IgG-RBCs for 10 minutes. LR and non-LR membrane fractions were pooled and immunoprecipitated with anti-BLT1 antibody. The immunoprecipitates were subjected to immunoblot, as in panel A. (F) Relative distribution of total BLT1, tyrosine-phosphorylated BLT1, FcγRI, and Src was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in (E), and expressed as LR/non-LR ratio of each. *P < .05 vs control, and #P < .05 vs IgG-RBC. (G) AMs were pretreated with PP2 or PP3, followed by incubation with (ii-iv) or without (i) 10:1 IgG-RBCs for 10 minutes. Cells were fixed and stained with CTxB-Alexa 488/anti-CTxB, Alexa 647 mouse anti-BLT1, and phycoerythrin mouse anti-FcγRI primary antibodies, and then visualized by confocal microcopy. Yellow patches signify areas of colocalization of FcγRI and GM1; purple patches, colocalization of FcγRI and BLT1; and white/gray patches, colocalized BLT1, FcγRI, and GM1 (the latter indicated by solid arrows). A dashed arrow pointing to a blue dot indicates BLT1 alone. Results are representative of at least 3 independent experiments. (H) AMs were pretreated for 5 minutes with vehicle control or PP2 or PP3 (100 nM), followed by 5-minute incubation with or without 10 nM LTB4. They were then incubated with IgG-RBCs (50:1), and phagocytic indices were determined as described in “Methods.” Phagocytosis is expressed as the percentage of the control value (IgG-RBCs alone) to which no drugs were added, and each bar represents the mean ± SEM from 3 individual experiments, each performed in quintuplicate. *P < .05 vs control, and #P < .05 vs LTB4.$

BLT1 phosphorylation by Src is required for IgG-induced FcγRI-BLT1 association in LRs. (A) AMs were incubated with or without 30:1 IgG-RBCs for 10 minutes and subjected to membrane fractionation. LR and non-LR membrane fractions were pooled and immunoprecipitated with an antibody against BLT1, after which immunoprecipitates were subjected to immunoblot analysis for the proteins shown on the right. (B) Relative distribution of total BLT1 as well as tyrosine- and serine-phosphorylated BLT1 was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel A, and is expressed as the LR/non-LR ratio of each. *P < .05 vs control. (C) AMs were pretreated with or without the Src kinase inhibitor PP2 (1 μM) or its negative control congener PP3 (1 μM) for 20 minutes, followed by incubation with or without 30:1 IgG-RBCs for 10 minutes. Pooled LR and non-LR fractions were prepared and subjected to immunoblot analysis for the proteins indicated at the right. (D) Relative distribution of BLT1 and FcγRI was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in panel C, and is expressed as the LR/non-LR ratio of each. *P < .05 vs control, and #P < .05 vs IgG-RBC. (E) AMs were incubated with or without PP2 or PP3 for 20 minutes, followed by challenge with 30:1 IgG-RBCs for 10 minutes. LR and non-LR membrane fractions were pooled and immunoprecipitated with anti-BLT1 antibody. The immunoprecipitates were subjected to immunoblot, as in panel A. (F) Relative distribution of total BLT1, tyrosine-phosphorylated BLT1, FcγRI, and Src was determined by densitometric analysis of immunoblots from 3 different experiments, as depicted in (E), and expressed as LR/non-LR ratio of each. *P < .05 vs control, and #P < .05 vs IgG-RBC. (G) AMs were pretreated with PP2 or PP3, followed by incubation with (ii-iv) or without (i) 10:1 IgG-RBCs for 10 minutes. Cells were fixed and stained with CTxB-Alexa 488/anti-CTxB, Alexa 647 mouse anti-BLT1, and phycoerythrin mouse anti-FcγRI primary antibodies, and then visualized by confocal microcopy. Yellow patches signify areas of colocalization of FcγRI and GM1; purple patches, colocalization of FcγRI and BLT1; and white/gray patches, colocalized BLT1, FcγRI, and GM1 (the latter indicated by solid arrows). A dashed arrow pointing to a blue dot indicates BLT1 alone. Results are representative of at least 3 independent experiments. (H) AMs were pretreated for 5 minutes with vehicle control or PP2 or PP3 (100 nM), followed by 5-minute incubation with or without 10 nM LTB₄. They were then incubated with IgG-RBCs (50:1), and phagocytic indices were determined as described in “Methods.” Phagocytosis is expressed as the percentage of the control value (IgG-RBCs alone) to which no drugs were added, and each bar represents the mean ± SEM from 3 individual experiments, each performed in quintuplicate. *P < .05 vs control, and #P < .05 vs LTB₄.

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