Blimp1

Human embryonic kidney (HEK) 293 cells were maintained in DMEM (Invitrogen). The media was supplemented with 10% FBS, 100 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. Polyclonal antibodies against the epitope tags (Flag, HA, and Myc), Ubiquitin, BLIMP1, and β-actin were obtained from Santa Cruz Biotechnology, Inc. Anti-Hrd1 and anti-Tubulin were purchased from Sigma-Aldrich. Fluorescence-labeled antibodies, including CD11c, CD11b, CD4, CD8, CD45.1, CD45.2, MHC-I, MHC-II, CD80, and CD86, were used for flow cytometry analysis (eBioscience). Hrd1 and the Ubiquitin expression plasmids were obtained as reported previously (Gao et al., 2008 (link)). Flag-BLIPM1 expression plasmids were purchased from Addgene. The truncation mutants of both Hrd1 and BLIMP1 were generated by PCR and subcloned into pCMV-Flag (Sigma-Aldrich) or pCMV-Myc vectors (Invitrogen).

Transient transfection was performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, in 60-mm dishes and using 2–3 µg of total DNA per transfection. 2 d after transfection, cells were lysed in 1× Nonidet P-40 lysis buffer and freshly added protease inhibitor cocktail. The cell lysates were mixed with antibodies (1 µg) for 2 h, followed by the addition of 30 µl of fast flow protein G–Sepharose beads (GE Healthcare) for an additional 2 h at 4°C. Immunoprecipitates were washed four times with Nonidet P-40 lysis buffer and boiled in 20 µl of 2× Laemmli’s buffer. Samples were subjected to 8–12% SDS-PAGE analysis and electro-transferred onto polyvinylidene difluoride membranes (Millipore). Membranes were probed with the indicated primary antibodies against Hrd1 (Sigma-Aldrich), BLIMP1 (Santa Cruz Biotechnology, Inc.), and Tubulin (Santa Cruz Biotechnology, Inc.), followed by horseradish peroxidase–conjugated secondary antibodies. Membranes were then washed and visualized with an enhanced chemiluminescence detection system (ECL; GE Healthcare). When necessary, membranes were stripped by incubation in stripping buffer (Bio-Rad Laboratories), washed, and then reprobed with other antibodies as indicated.