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. 2014 Nov 17;211(12):2467–2479. doi: 10.1084/jem.20140283

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© 2014 Yang et al.

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Figure 6. — Hrd1 interacts with BLIMP1. (A) A Myc-Hrd1 expression plasmid was transfected with or without Flag-BLIMP1 expression plasmid into HEK293 cells. Expression of Hrd1 and BLIMP1 in whole cell lysates was confirmed by immunoblotting (lanes 1 and 2). Hrd1 interaction with BLIMP1 was determined by immunoprecipitation (IP) with anti-Flag antibody followed by immunoblotting with anti-Myc antibody (top, lanes 3 and 4). The same membrane was reprobed with anti-Flag antibody to confirm BLIMP1 expression (bottom). (B) Interaction between endogenous Hrd1 and BLIMP1 in mouse BMDCs was analyzed by IP with anti-BLIMP1 using normal rabbit IgG as a control, followed by immunoblotting with anti-Hrd1 (top). The same membrane was reprobed with anti-BLIMP1 (bottom). (C) BMDCs were stimulated with different doses of LPS as indicated for 2 h. The interaction between BLIMP1 and Hrd1 was determined as in B. (D and E) Truncation mutants of Hrd1 were generated (D) and their interactions with BLIMP1 in transiently transfected HEK293 cells were determined by IP and immunoblotting (E) as described in A. (F and G) Truncation mutants of BLIMP1 were generated (F) and their interactions with full-length Hrd1 in the transiently transfected HEK293 cells were determined by IP and immunoblotting (G) as described in A. TM, transmembrane; Pro, proline; AD, acidic domain; PR, positive regulatory; ZF, zinc finger.