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. 2008 Jun 9;205(6):1357–1368. doi: 10.1084/jem.20070950

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© 2008 Aoufouchi et al.

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Figure 6. — AID is ubiquitinated in the nucleus of activated mouse B splenocytes. (A) Purified B cells from mouse spleen were activated by incubation with LPS and IL-4. At the indicated days after activation, cells were collected and total (T), cytoplasmic (C), or nuclear (N) protein extracts were prepared as described in Materials and methods and analyzed by immunoblotting, with a monoclonal anti–mouse AID antibody. Antinucleolin antibody was used as a nuclear protein control. (B) At day 4, activated B cells were treated with LMB and/or MG132, as indicated. SDS-PAGE–fractionated extracts were probed with anti–mouse AID antibody. Actin was used as loading control. (C) At day 2 of IL-4/LPS activation, 2 × 10⁷ cells were transfected with Ub-HA–expressing vector using Amaxa nucleofection, as indicated in Materials and methods, and either left untreated or incubated for 4 h with both MG132 and LMB, 16 h after transfection. After treatment, cell lysates were denatured and immunoprecipitated with agarose-conjugated anti-HA antibodies before Western blot analysis with anti-AID monoclonal antibodies. MW markers and migration positions of endogenous AID are indicated.