The LUBAC contributes to aberrant activation of NF-κB and survival of ABC DLBCL lines. (A) Cell lysates from ABC DLBCL lines (OCI-Ly3 and OCI-Ly10) and from GCB DLBCL lines (OCI-Ly7 and OCI-Ly19) were IP with antibodies to SHARPIN, and IBs were performed as indicated. The asterisk indicates nonspecific bands. (B) DLBCL lines were infected with a retrovirus that expressed a control nonspecific (NS^sh) or a HOIP^sh shRNA together with GFP. Shown is the fraction of GFP-positive cells over time relative to day 4 postinfection. (C) Lysates from OCI-Ly3, OCI-Ly10, OCI-Ly19, and BJAB cells transfected with control NS siRNA or with siRNA against the LUBAC (HOIP plus HOIL-1 plus SHARPIN) for 48 hours were analyzed by IB as indicated. (D-E) DLBCL lines were transfected as in (C) for 72 hours and stained with DiOC₆ and analyzed by flow cytometry. Histograms in (D) represent the fold of NS-treated sample. Shown is the mean ± SEM from 3 independent experiments (ns, **P < .001; ****P < .0001 by ANOVA). (F) IκBα levels were assessed by IB 72 and 96 hours posttransfection in lysates as in (C). (G-H) ABC DLBCL lines (OCI-Ly3 and OCI-Ly10) and GCB DLBCL lines (OCI-Ly19 and BJAB) were transfected as in (C) for 72 hours. NF-κB p65 subcellular location was examined by confocal microscopy. Nuclei were illuminated with 4′,6-diamidino-2-phenylindole (DAPI). Histograms in (H) show the mean ± SEM from 3 independent experiments (ns, ****P < .0001 by ANOVA). Data are representative of 3 independent experiments.