DYRK1a phosphorylates TRAF3. (A-B) In vitro kinase assays using HA-DYRK1a isolated by IP from transfected 293 cells and the indicated human (A) or murine (B) GST-TRAF3 recombinant proteins as substrates. The kinase assay membrane was subjected to IB to detect HA-DYRK1a and the GST-TRAF3 substrates. (C) In vitro kinase assays using endogenous DYRK1a isolated by IP from BAFF- or CD40L-stimulated WT B cells and murine GST-TRAF3 substrate. (D) Chimeric mice were generated by adoptively transferring Rag1^−/− mice with Traf3^BKO bone marrow cells transduced with expression vectors for WT TRAF3 or T29A mutant. Immunoblot was performed to analyze the indicated proteins using cytoplasmic or nuclear extracts of BAFF-stimulated splenic B cells isolated from chimeric mice of Traf3^BKO bone marrow reconstituted with TRAF3 WT or T29A. (E) Flow cytometric analysis of the B220⁺ B cells and TCRβ⁺ T cells in the spleen (SP) or mesenteric lymph nodes (mLN) of the indicated chimeric mice described in panel D. (F) Flow cytometric analysis of the mature (M; B220⁺CD93⁻), immature (Imm; B220⁺CD93⁺), follicular (FO; B220⁺CD21^intCD23⁺), and marginal zone (MZ; B220⁺CD21^hiCD23⁻) B cells in the spleen of the indicated chimeric mice described in D. Data are presented as a representative plot, and summary graphs are mean ± SD values based on multiple mice, and P values are determined by an unpaired, 2-tailed Student t test. *P < .05; **P < .01; ***P < .001.