DYRK1a mediates noncanonical NF-κB activation and B-cell survival. (A-C) B cells purified from the spleen of WT and Dyrk1a^BKO mice were cultured for 48 h in 96-well plate (2 × 10⁵ cells per well), in the presence of medium control, BAFF or CD40L. The cells were subjected to flow cytometry to quantity dead and viable cell populations, presented as a representative plot (A) and summary graphs based on multiple mice (B), or proliferation assays based on ³H-thymidine incorporation (C). (D) Immunoblot analysis of the indicated proteins using cytoplasmic (Cyt) or nuclear (Nucl) extracts of BAFF-stimulated WT and Dyrk1a^BKO splenic B cells. (E-F) Immunoblot analysis of the indicated proteins in whole-cell extracts of BAFF-stimulated WT and Dyrk1a^BKO splenic B cells. Data are presented as a representative blot (E) and a summary graph of densitometric quantification of the NIK protein bands based on 3 independent experiments (F). NIK level was quantified as ratio to β-Actin and presented as fold to the nontreated (NT) lane (set as 1). (G) qRT-PCR analysis of Map3k14 mRNA in WT or Dyrk1a^BKO splenic B cells stimulated with BAFF for 16 h. (H) DYRK1a was immunoprecipitated from BAFF- or CD40L-stimulated WT B cells and subjected to immunoblot assays using anti-phosphorylated tyrosine (p-Tyr) or DYRK1a. Summary graphs are presented as mean ± SD, and P values were determined by an unpaired, 2-tailed Student t test (B-C,F). *P < .05; **P < .01; ***P < .001.