![Figure 4. Phosphorylation of p85 and measurement of p110 activity. A concentration of 10–7 M chemoattractant was chosen. (A) Cells in control conditions. Top subpanel: Neutrophil suspensions were stimulated with or without 100 nM fMLP or 100 nM IL-8 for 60 seconds as indicated. The p85α was immunoprecipitated by means of a polyclonal antibody against p85α, and processed for immunoblotting (IB) as detailed in “Materials and methods” with p85α (anti-p85α), phosphorylated p85 (anti–pp85), or antiphosphotyrosine (anti-PY) antibodies. The data shown are representatives of at least 3 experiments, with identical results, on separate cell preparations. Bottom subpanel: In vitro measurement of PtdIns 3-kinase activity in neutrophils. The cells were activated with 100 nM fMLP (•) or 100 nM IL-8 (○) for the indicated periods of time. Then, p85 was coimmunoprecipitated with p110 from the activated cellular lystates, and p110 kinase activity was measured by [32P] incorporation into PtdIns substrate. The panels above the graph depict the result of a representative TLC plate, and the average densitometric values as a percentage of control ± SD shown are calculated from 3 independent experiments. (B) Same as panel A for cells pretreated for 30 minutes with 30 pM GM-CSF. (C) Same as panel A for cells pretreated for 30 minutes with 320 μU/mL insulin.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/102/6/10.1182_blood-2002-09-2936/6/h81834950004.jpeg?Expires=1722731281&Signature=tWHEbEbiUw09BGUOl027p~QvW9NawLLrkfJIfasY7lQHgqdh7xTXY6lPJEnBV7aqdg2HxjYzfP2DExwYITyCS1rG-j4AHpWDmiyuTqC29GeM8PLT5HvgLiW9zu7aQ8fnFlzjn3ikAVsrEgME4jSnoD~iIza-U5RW7hyHIhi5XKv336SqG-CtTtL1pnhSD8070zMJ6pnvfzAggje4atuNwT~l-DVfL8T~umOQLd~4lpJckvBvczBwyU8ZYczACJxlJL8XhGfjwPfU19EzeSKOsHIe3QTSOluqs~TmYTyeL~wvTGOpqTq4Iqz2hc9ts95xueVerxN5QANigdq5-oHjSg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Phosphorylation of p85 and measurement of p110 activity. A concentration of 10–7 M chemoattractant was chosen. (A) Cells in control conditions. Top subpanel: Neutrophil suspensions were stimulated with or without 100 nM fMLP or 100 nM IL-8 for 60 seconds as indicated. The p85α was immunoprecipitated by means of a polyclonal antibody against p85α, and processed for immunoblotting (IB) as detailed in “Materials and methods” with p85α (anti-p85α), phosphorylated p85 (anti–pp85), or antiphosphotyrosine (anti-PY) antibodies. The data shown are representatives of at least 3 experiments, with identical results, on separate cell preparations. Bottom subpanel: In vitro measurement of PtdIns 3-kinase activity in neutrophils. The cells were activated with 100 nM fMLP (•) or 100 nM IL-8 (○) for the indicated periods of time. Then, p85 was coimmunoprecipitated with p110 from the activated cellular lystates, and p110 kinase activity was measured by [32P] incorporation into PtdIns substrate. The panels above the graph depict the result of a representative TLC plate, and the average densitometric values as a percentage of control ± SD shown are calculated from 3 independent experiments. (B) Same as panel A for cells pretreated for 30 minutes with 30 pM GM-CSF. (C) Same as panel A for cells pretreated for 30 minutes with 320 μU/mL insulin.
