Fra 2

Dynabeads Protein G (Thermo Fisher Scientific) was incubated with 50 µg Fra-2 (Santa Cruz Biotechnology, Inc.) for 10 min at 22°C. Afterward, immunoprecipitation assay was performed with proteins from bone marrow B220⁺ B cells according to the manufacturer’s protocol. In brief, 100 µl of protein solution was incubated for 30 min with the complex Dynabeads Protein G-Fra-2 antibody. The target antigen was eluted under nondenaturing conditions, added 20 ml Elution Buffer (Thermo Fisher Scientific), and incubated with rotation for 2 min at 22°C to dissociate the complex. The eluted protein was stored at −80°C until Western blot analysis.

ChIP experiments were performed with ChIP-IT Express kit (Active Motif) according to the manufacturer’s protocol. 14 µl FosB, c-Fos, Fra-1, Fra-2, JunB, JunD antibodies (Santa Cruz Biotechnology, Inc.), Irf4 (MUM1; Abcam), cJun, and Foxo1 (Cell Signaling Technology) as well as a rabbit control IgG were used for the immunoprecipitation.