PTEN inhibitor SF1670 enhances fMLP-induced PtdIns(3,4,5)P3 signaling in both human and mouse neutrophils. (A) Human primary neutrophils were pretreated with 500nM SF1670 at 37°C for 30 minutes and then stimulated with 100nM fMLP for indicated times. The levels of phosphorylated AKT, p38, and c-Jun NH₂-terminal kinase (JNK) were assessed by Western blotting with specific antibodies. (B) Human neutrophils were incubated with indicated concentrations of SF1670 at 37°C for 30 minutes and then stimulated with or without 100nM fMLP for 4 minutes. Total and phosphorylated AKT levels were assessed as described in panel A. (C) Human neutrophils were incubated with 500nM SF1670 at 37°C for 30 minutes and then washed twice with PBS. After being incubated in PBS + 2% BSA for indicated times, cells were stimulated with 100nM fMLP for 4 minutes, and then the levels of total and phosphorylated AKT were assessed by Western blotting using specific antibodies. (D) PTEN inhibitor SF1670 enhanced fMLP-induced PtdIns(3,4,5)P3 signaling in mouse neutrophils. Mouse bone marrow derived neutrophils were purified by negative selection using an EasySep system (StemCell Technologies). Cells were pretreated with 500nM SF1670 at 37°C for 30 minutes and then stimulated with 500nM fMLP for indicated times. The levels of phosphorylated AKT were assessed by Western blotting with specific antibody. (E) PtdIns(3,4,5)P3 level increased in SF1670-treated mouse neutrophils. Cellular PtdIns(3,4,5)P3 levels were measured using a mass ELISA kit (Echelon). In brief, PtdIns(3,4,5)P3 was extracted from 5 million purified mouse bone marrow–derived neutrophils following the manufacturer's lipid extraction protocol. The samples were mixed and incubated with a PtdIns(3,4,5)P3 detector protein and then added to a PtdIns(3,4,5)P3-coated microplate for competitive binding. A peroxidase-linked secondary detector and colorimetric detection were used to quantify the PtdIns(3,4,5)P3 detector protein bound to the plate. The colorimetric signal is inversely proportional to the amount of PtdIns(3,4,5)P3 in the sample. (F) Bone marrow–derived neutrophils isolated from wild-type (WT) and myeloid specific PTEN knockout mice²⁸  were preincubated with or without 500nM SF1670 at 37°C for 30 minutes. Cells were then stimulated with or without 1 μM of fMLP for 4 minutes. Total and phosphorylated AKT levels were measured as described in panel A.