Nuclear BAFF-R interacts with IKKβ and functions in histone H3 phosphorylation. (A) FLAG-BAFF-R fusion protein was purified by FLAG antibody from a nuclear extract of a stably transfected NHL-B-cell line expressing FLAG-BAFF-R. Extract samples were probed with antibodies against CBP, IKKα/β, BAFF-R, or FLAG in WB (left panel). Wild-type BAFF-R was immunoprecipitated (IP) from the nuclear extract using BAFF-R antibody and probed with IKKβ, BAFF-R, or histone H3 antibody on a Western blot (middle and right panels). Lamin B (a nuclear marker), syndecan 4 (a plasma membrane marker), and calreticulin (an endoplasmic reticulum marker) were used as loading controls. (B) Histone H3, CBP, or IKKβ protein in NHL-B cells was identified using Cy2 (green), and BAFF-R protein was identified using Cy3 for confocal microscopic analysis. (C) Histone H3 or IKKβ protein was stained with Cy2, and BAFF-R protein was stained with Cy3 in normal peripheral blood B cells stimulated with anti-IgM and BLyS. Samples were analyzed by confocal microscope. (D) IKKβ protein was stained with Cy2 fluorescence, BAFF-R protein was stained with Cy3, and TOPRO-3 was used as a nuclear marker. (E) Nuclear extracts from NHL-B cells transfected with an empty vector, a BAFF-R expression plasmid, or a BAFF-R NLS-mutant expression plasmid were probed with BAFF-R, phosphorylated histone H3 (Ser10), or Oct-1 (nuclear-protein loading control) antibody in WB (left panel). Whole-cell extracts from NHL-B cells treated with specific BAFF-R siRNA or scrambled control siRNA (control) were probed with BAFF-R, phosphorylated histone H3, or actin antibody in WB (right panel). (F) Recombinant histone H3 was incubated, with or without a FLAG-BAFF-R complex purified by IP with FLAG antibody from NHL-B cells (Jeko) expressing FLAG-BAFF-R protein in an in vitro kinase assay. IgG was used as an IP control. ³²P-labeled phosphorylated H3 protein was detected by gel electrophoresis. Unphosphorylated histone H3 (free histone H3) in phosphorylation reaction was detected by WB. (G) Recombinant H3 was incubated with recombinant IKKα or IKKβ, with or without BAFF-R, in an in vitro kinase assay. ³²P-labeled phosphorylated H3 was detected by gel electrophoresis. BAFF-R and free histone H3 were detected by WB.