CARD11 regulates c-Jun and JunB level in primary B cells and DLBCL cells. (A) Murine primary B cells were isolated from spleens of wt or CARMA1-deficient mice. The cells (4 × 106 cells per sample) were stimulated with phorbol myristate acetate (PMA) plus ionomycin (P/I; 20 and 100 ng/mL, respectively) for various time points. Whole-cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by western blotting using antibodies against c-Jun, JunB, JNK2, or β-actin. (B) Cell lysates were prepared from the indicated lymphoma cell lines and subjected to SDS-PAGE followed by western blotting. (C) Nuclear extracts were prepared from DLBCL cells and analyzed by electrophoretic mobility shift assay (EMSA) by using 32P-labeled probes containing AP-1– or Oct-1–binding sites. (D) For the supershift assay, nuclear extracts were pre-incubated (30 minutes at 4°C) with the indicated antibodies and then subjected to EMSA. (E) c-Jun ubiquitination in lymphoma cell lines. The cells were stimulated with P/I for 5 minutes, and the cell lysates were precipitated with c-Jun antibodies for 16 hours. The immunocomplexes (top panel) and whole-cell lysates (bottom panel) were subjected to SDS-PAGE and analyzed by western blotting by using ubiquitin or c-Jun antibodies. (F-G) OCI-Ly7 cells were stably transfected withCARD11wt, constitutively active CARD11del (the linker region between the coiled-coil and PDZ domains of CARD11 was deleted) or CARD11(L244P) mutant by lentiviral infection. Whole-cell lysates were subjected to SDS-PAGE followed by western blot analysis using the indicated antibodies. (H) OCI-Ly7 cells or cells transduced with the indicated CARD11 constructs were subjected to EMSA to determine AP-1 binding activity. Exp, exposure; IB, immunoblotting; IgG, heavy chain; IP, immunoprecipitation; ph, phosphorylated; Ub, ubiquitin; WB, western blotting; *, shifted band.
