Optimal BAFF induction of Mcl1 and A1 expression requires ERK5. (A and B) Purified splenic FM B cells from mb1-cre⁺ erk5^fl/fl mice and mb1-cre⁺ erk5^wt/wt control mice were cultured with BAFF or control medium (-) for 16 h (A) or 1 h (B). Whole-cell lysates were immunoblotted for the indicated proteins. Results are representative of three independent experiments (3 mice/genotype each). In A, graphs represent mean band intensities for the indicated proteins (±SEM; n ≥ 5/genotype each), normalized to tubulin loading control. In B, quantitation of band intensities (6 mice/genotype) demonstrated that ERK5 deficiency did not significantly alter P-p105 levels in BAFF-stimulated cells (data not shown). (C) mRNA levels of the indicated genes from rested purified splenic FM B cells from mb1-cre⁺ erk5^fl/fl mice and mb1-cre⁺ erk5^wt/wt control mice were determined by quantitative RT-PCR. Data were normalized to Hprt1 mRNA and are represented as mean fold change (±SEM) relative to unstimulated WT cells. (D) Purified splenic FM B cells from the indicated mouse strains were cultured in duplicate with BAFF for 16 h and mRNA expression was assessed as indicated in C. (E) Purified splenic FM B cells from the indicated mouse strains were cultured for 48 h with BAFF or control medium (-) plus PI-103 (PI; 0.5 µM), GDC-0941 (GDC; 0.5 µM), or vehicle control (DMSO). The fraction of live FM B cells was determined by flow cytometric analysis of 7AAD staining (mean ± SEM; triplicates). In C–E, results are representative of three separate experiments (three mice/genotype each). (F) WT mice were injected intraperitoneally twice (day 1 and 3) with 100 µg anti-BAFF antibody or isotype control. At day 4, splenic FM B cells were purified and cell lysates immunoblotted. Results are representative of two independent experiments (4 mice/genotype each). Graphs (right) represent mean band intensity (±SEM; n = 7/genotype each), normalized to tubulin loading control. (G) mb1-cre⁺ erk5^fl/fl mice and mb1-cre⁺ erk5^wt/wt control mice were injected intraperitoneally with BrdU daily for 5 days. Spleens were harvested at day 6 and BrdU⁺ cells assayed by flow cytometry. Results are representative of two independent experiments (five mice/genotype each). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.