PI3Ks are activated by p38 downstream of TNFR1. (A) p38 and PI3Ks are activated downstream of TNFR1. Neutrophils were kept untreated (Control, Medium) or incubated with 10 μg/mL of anti-TNFR1 or anti-TNFR2 antibodies for 90 minutes. Cells were subsequently stimulated with 50 ng/mL of TNF-α for 15 minutes and cell lysates were analyzed as in Figure 3C. A representative immunoblot is shown, and protein expression levels were quantified relative to the control condition (n = 3). (B) Neither class IA PI3Ks nor p38 is associated with activated TNFR1. Human neutrophils were stimulated with 20 ng/mL of TNF-α for 5 minutes. The lysates were incubated with anti-TNFR1 antibody (H398) or IgG2a control antibody. Immunoblots were probed for p38, the PI3K class IA regulatory subunits p85α/p85β (p85), TRADD, and GAPDH (n = 3). IP indicates immunoprecipitation. (C) p38 is upstream of PI3K activation. Neutrophils were treated for 90 minutes with vehicle (Control, Medium), with the PI3K inhibitors wortmannin (100nM) and PI103 (100nM), or with the p38 inhibitors SB203580 (1μM) and PD169316 (10μM) before TNF-α (50 ng/mL) stimulation. Immunoblots were probed for phosphorylated Thr308 and Ser473 AKT, phosphorylated Thr180 and Tyr182 p38, phosphorylated Thr334 MK2, and GAPDH. A representative immunoblot is shown. Protein expression levels were quantified relative to the control condition (n ≥ 3). (D) Phosphorylation of p38 precedes phosphorylation of AKT. Neutrophils were stimulated for the indicated times with 50 ng/mL of TNF-α. Cell lysates were analyzed by immunoblotting for phosphorylated Ser473 AKT or phosphorylated Thr180/Tyr182 p38. GAPDH protein levels were analyzed as loading controls. A representative immunoblot is shown. Protein expression levels were quantified relative to the control condition (n = 3).