The LUBAC binds to CBM and IKK complexes and participates in antigen receptor-mediated NF-κB activation. (A) Jurkat T lymphocytes were stimulated with 20 ng.mL⁻¹ PMA plus 300 ng.mL⁻¹ ionomycin (P/I) or with 1 μg.mL⁻¹ anti-CD3 and anti-CD28 (CD3/28) for 0, 10, and 20 minutes. Cell extracts were prepared and immunoprecipitated (IP) with antibodies to CK1α, BCL10, SHARPIN, or NEMO, and immunoblots (IBs) were performed as indicated. Closed circle and asterisk indicate the protein of interest and nonspecific bands, respectively. Ub, ubiquitin. Molecular weight markers (kDa) are indicated. (B) Jurkat cells were transfected with a nonspecific control siRNA (NS) or with 3 individual siRNAs against HOIP (HOIP.1-3). Three days later, cells were transfected with pNF-κB-Luc reporter and pTKRL control plasmids for an additional 24 hours, prior stimulation with 0.5 μg.mL⁻¹ anti-CD3 and anti-CD28 (3/28), or with P/I as in (A), or with 10 ng.mL⁻¹ TNFα for 6 hours. Histograms represent the mean ± standard error of the mean (SEM) of 3 independent experiments. **P < .001; ****P < .0001 compared with cells transfected with NS siRNA (analysis of variance [ANOVA]). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, nonsignificant; RLU, relative light unit; Unst, unstimulated cells. Inset IB shows the level of protein knockdown. (C) NS- and HOIP-silenced Jurkat T cells were stimulated with 1 µg.mL⁻¹ anti-CD3 and anti-CD28 (CD3/28) for 0, 30, and 60 minutes. Nuclear and cytosolic fractions were purified, and IBs were performed as indicated. Tubulin and poly (ADP-ribose) polymerase (PARP) served as loading controls. (D-E) Confocal microscopy micrographs of NF-κB p65 (green) in Jurkat T cells transfected with an NS or HOIP siRNA and stimulated with 1 µg.mL⁻¹ anti-CD3 and anti-CD28 (CD3/28) for 1 hour. Nuclei were also stained with 4′,6-diamidino-2-phenylindole (in blue). In (E), the percentage of cells with nuclear p65 was calculated by scoring >150 cells for each sample. Shown is the mean ± SEM from 3 independent experiments (****P < .0001 by ANOVA). (F) NS- or HOIP-silenced human peripheral blood mononuclear cells were stimulated with 1 and 100 ng.mL⁻¹ anti-CD3 and anti-CD28 for 16 hours. Interleukin-2 secretion in the culture supernatants was determined by enzyme-linked immunosorbent assay. Histograms represent the mean ± SEM of 3 independent experiments (****P < .0001 by ANOVA). Inset immunoblots show the knockdown efficiency. (G) Cell extracts from NS- and HOIP-silenced Jurkat cells stimulated as in (C) for 0, 10, 20, and 30 minutes were analyzed by IB. (H) NS- and HOIP-silenced BJAB B cells were stimulated with 10 ng.mL⁻¹ PMA plus 300 ng.mL⁻¹ ionomycin (P/I) for 0, 10, 20, and 30 minutes. Cell lysates were prepared and analyzed by IB as indicated. (I) NEMO binding to HOIP, BCL10, and MALT1 was assessed by IP/IB in NS- and HOIP-silenced Jurkat cells stimulated with P/I as in (A). Lys., lysate. (J) Cell extracts from Jurkat cells stimulated with (P/I) as in (A) or with 10 ng.mL⁻¹ TNFα for 0, 10, and 20 min were IP with an antibody against M1-linked Ub. IBs were performed as indicated. The asterisk shows the anti-M1-Ub. (K) Jurkat cells were retrovirally infected to express GFP, GFP plus an shRNA against human HOIP (HOIP^sh), RNAi-resistant HOIP wild type plus HOIP^sh, and catalytically inactive HOIP (HOIP-CS) plus HOIP^sh. Cells were stimulated as in (J) for 30 minutes and analyzed as in (E). Shown is the mean ± SEM from 3 independent experiments (ns, ***P < .001; ****P < .0001 by ANOVA). Cell lysates were prepared and IB performed as indicated. Molecular weight markers (kDa) are shown. Data are representative of 2 (I) or at least 3 independent experiments (A-H, J-K).