Fig. 5

C. tropicalis induced Syk-mediated PKM2 Tyr105 phosphorylation and PKM2 nuclear translocation in MDSCs. A WT and Clec4d^−/− MDSCs were stimulated with heat-inactivated C.tropicalis (MOI = 2) for 24 h. Cell lysates were analyzed by immunoblotting for phosphorylated (p-) and total Syk. B Schematic representation showing the substrate phosphorylation sites of Syk. C List showing eight putative phosphorylated tyrosine sites of PKM2. D Schematic representation showing posttranslational modification (PTM) sites of PKM2. Data of B-D was from PhosphoSitePlus® (http://www.phosphosite.org/) [32]. E and F WT and Clec4d^−/− MDSCs were stimulated with heat-inactivated C.tropicalis (MOI = 2) for 6 or 24 h. Cell lysates were analyzed by immunoblotting for PKM2 (p-Y105) (E). Nuclear extracts were analyzed by immunoblotting for PKM2 (F). G WT MDSCs were stimulated with heat-inactivated C.tropicalis (MOI = 2) for 18 h. Immunoprecipitation was carried out with antibodies against PKM2 or rabbit IgG. The immunoprecipitates and cell lysates were analyzed by immunoblotting for the indicated proteins. H and I WT MDSCs were stimulated with heat-inactivated C.tropicalis (MOI = 2) in the presence or absence of Syk inhibitor R406 (as indicated concentration) for 18 or 24 h. Cell lysates were analyzed by immunoblotting for PKM2 (p-Y105) (H). Nuclear extracts were analyzed by immunoblotting for PKM2 (I). The results shown here are expressed as the mean ± SEM. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. The following statistical analyses were performed: unpaired Student’s t-test or one-way ANOVA where appropriate