Endogenous Nur77 binds endogenous Bcl-B in cells of plasma cell lineage. (A) HEK293T cells were transfected with 1 μg plasmids encoding Myc-tagged Bcl-B, Bcl-2, Bcl-X_L, Bfl-1, Mcl-1, and Bcl-W proteins. After 24 hours, cells were lysed and analyzed by SDS-PAGE/immunoblotting using anti-Myc (top blot) and anti–Bcl-B (bottom blot) antibodies. Molecular weight markers are shown in kilodaltons (kDa). (B) Immunohistochemical detection of Bcl-B in normal human bone marrow. Serial sections of bone marrow biopsy were stained with (i) preimmune serum, (ii) anti–Bcl-B antiserum, (iii) anti–Bcl-B antiserum preadsorbed with GST–Bcl-B, and (iv) anti–Bcl-B serum preadsorbed with Bcl-W peptide. Specimens were counterstained with nuclear red dye. Original magnifications are × 1000. (C) Lysates from various myeloma and nonmyeloma cell lines were analyzed by SDS-PAGE/immunoblotting using Bcl-B, Bcl-2, and tubulin antibodies. (D) RPMI 8226 cells were stimulated for 1, 3, or 5 hours with TPA (100 ng/mL) and ionomycin (1 μM). Lysates were prepared, and immunoprecipitation was performed with monoclonal anti-Nur77 monoclonal antibody or mouse IgG. Immunoprecipitates and lysates were analyzed by SDS-PAGE/immunobloting using anti-Nur77 (top blot) or anti–Bcl-B (bottom blot) antibodies. In panels C and D, “293/Bcl-B” represents lysate (20 μg) from HEK 293T cells transfected with plasmid encoding Bcl-B protein, used as a positive control. Asterisk indicates a nonspecific band. Phospho form of Nur77 is indicated by the circled P.