PKM2 mediates poststroke inflammation by promoting phosphorylation of STAT3 in neutrophils. (A) Left: representative image of flow cytometric analysis for MPO from each group. Right: quantification of MPO expression levels in peripheral neutrophils isolated 6 hours post-reperfusion in mice with stroke. Neutrophils were stimulated with a suboptimal concentration of PMA (10 ng/mL). (B) Elastase levels as determined by ELISA from the cell extracts of peripheral neutrophils, isolated 6 hours post-reperfusion in mice with sham-surgery or stroke. (C) Left: representative immunostained images for neutrophils (brown Ly6B.2-positive cells indicated by red arrows) in infarcted brain regions. Inset in the boxed region is magnified and shown in microphotograph. Scale bar, 100 μm. Right: quantification. (D) Peripheral neutrophils were isolated 6 hours postreperfusion in mice with stroke, and cell extracts were IP with PKM2 antibody or control IgG and immunoblotted for PKM2 and STAT3. (E) Western blot analysis of PKM2 cell extracts from the peripheral neutrophils isolated 6 hours post-reperfusion in mice with stroke. The quantitative phospho STAT3 and phospho NF-κβ intensity (normalized to the total STAT3 and NF-κβ, respectively) are shown on the right. Data are from female mice and are mean ± SEM. n = 5 (A); n = 3-4 (B-C,E). Data are from 2-way repeated measures ANOVA (Kruskal-Wallis test) followed by Fisher’s LSD test (A-B) and unpaired Student t-tests (C,E). FSC, forward scatter; IgG, immunoglobulin G; IP, immunoprecipitated; MPO, myeloperoxidase; MFI, mean fluorescence intensity.