Fig. 1
Stimulation of IgSF11 induces the phosphorylation of pyruvate kinase M2. a Schematics of IgSF11 (top), the human CD3 extracellular-transmembrane region fused to the full IgSF11 intracellular region (hCD3-iFL) (middle), and the IgSF11-Mt353 intracellular region (hCD3-iMt353) (bottom) chimeric proteins. The hCD3-iFL and hCD3-iMt353 mutants were used for retroviral transduction of IgSF11−/− BMMs. All constructs were Flag-tagged at the C-terminus. TM; transmembrane domain, PB; PDZ binding domain. b IgSF11−/− BMMs were retrovirally transduced with the indicated vectors, cultured with M-CSF + RANKL in the presence of anti-hCD3 antibody or control IgG and stained for TRAP. The frequency of TRAP+ multinucleated cells (3 nuclei or more per cell) is shown. Scale bars represent 100 μm. EV; empty vector. The data are presented as the means ± S.D. Each dot represents a technical replicate. c Tyrosine phosphorylation downstream of IgSF11. IgSF11−/− BMMs were retrovirally transduced with the indicated vectors, cultured with M-CSF + RANKL for two days and then stimulated with anti-hCD3 antibodies for the indicated times. IgSF11+/+ BMMs that were transduced with an empty vector were used as a negative control. Western blotting was performed with the indicated antibodies. d IgSF11−/− BMMs were retrovirally transduced with hCD3-iFL, cultured with M-CSF + RANKL for two days and then stimulated with anti-hCD3 antibodies for 30 min. Whole-cell lysates were passed through phosphoprotein affinity columns. The eluted fraction was separated by SDS–PAGE, visualized with Coomassie blue R-250 and/or immunoblotted with 4G10. The arrow indicates the protein band that was subjected to mass spectrometry. e IgSF11 induces the phosphorylation of PKM2. IgSF11−/− BMMs were retrovirally transduced with the indicated vectors, cultured with M-CSF + RANKL for two days and then stimulated with anti-hCD3 antibodies for the indicated times. Western blotting was performed with the indicated antibodies. The results are representative of at least three independent experiments
