ATF3 is overexpressed and forms heterodimers with Jun subunits in ABC DLBCL cell lines. (A) Analysis of ATF3 and JDP2 protein expression in GCB and ABC cell lines was determined by western blot using ATF3 and JDP2 antibodies. Filled arrowhead indicates the position of phosphorylated JDP2; open arrowheads indicate nonphosphorylated JDP2. (B) Immunoblot analysis of lysates of HBL-1 (ABC DLBCL) and BJAB (GCB DLBCL) cell lines, transduced with control shRNA or with CARMA1-, MALT1-, IRAK1-, or MyD88-specific shRNA. Silencing efficiency was assessed by western blot analysis using anti-CARMA1, anti-MALT1, anti-IRAK1, and anti-MyD88 antibodies. ATF2, ATF3, ATF7 protein levels were assessed by western blot. Blotting for tubulin served as loading control in panels A and B. (C) Lysates of indicated ABC and GCB cell lines were treated with the cross-linker BS3 or with solvent alone for 1 hour at 4°C. Cross-linked (open arrowheads) and non–cross-linked (filled arrowheads) proteins were assessed by western blot using anti-ATF3 antibodies. White asterisk indicates the position of a nonspecific band detected by anti-ATF3. (D) ABC DLBCL cell lines were immunoprecipitated with anti-c-Jun, anti-JunB, anti-JunD, or anti-IRE1α antibodies (Ctr, control antibody). Immunoprecipitated proteins (IPs) were assessed with anti-c-Jun, anti-JunB, and anti-JunD antibodies, and coprecipitating proteins (co-IP) detected by anti-ATF3. *Heavy chain or light chain of the c-Jun, JunB, JunD, and control antibodies; filled arrowhead indicates ATF3 in the co-IP. (E) The composition of protein complexes of ATF3 with Jun family members (type 2 complexes) is depicted. Data are representative of 3 (A) and 2 (B-D) independent experiments.