BAFF-induced FM B cell survival requires ERK5. (A) Purified splenic FM B cells from mb1-cre⁺ erk5^fl/fl mice and mb1-cre⁺ erk5^wt/wt control mice were cultured for 48 h with BAFF, α-IgM, BAFF plus α-IgM, or control medium (-). The fraction of live FM B cells was determined by flow cytometric analysis of 7AAD staining (mean ± SEM; n = 3 mice/genotype; triplicate cultures). Results are representative of at least three separate experiments. (B) BAFF-R protein levels on gated AA4.1^-B220⁺IgM⁺CD23⁺ FM B cells of the indicated genotypes were analyzed directly ex vivo by flow cytometry. T cells from mb1-cre⁺ erk5^wt/wt mice were used as a negative control. (C) Purified splenic FM B cells were cultured for 16 h ± BAFF. Immunoprecipitated ERK5 from whole-cell lysates was incubated ± λ phosphatase and analyzed by immunoblotting. (D and E) Pooled WT purified splenic FM B cells were cultured for 16 h with BAFF or control medium (-) plus vehicle control (DMSO), 10 µM BIX02188, 10 µM BIX02189, or 2 µM PD184352 (PD). Cell lysates were immunoblotted. (F) Total lysates of purified splenic FM B cells stimulated with α-IgM were immunoblotted. (G) Pooled WT purified splenic FM B cells were cultured for 16 h with BAFF or control medium (-) plus BAFF-R blocking antibody or isotype control. Total cell lysates were immunoblotted. All results are representative of two or more independent experiments. ****, P < 0.0001.