PKK regulates NF-κB activation induced by BAFF. (A) Surface expression of the indicated BAFF receptors in SUDHL-6 cells. SUDHL-6 cells were incubated with antibodies specific for human BAFF receptor (BR3 or BAFF-R), B cell–maturation antigen (BCMA), or transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) (filled histograms) or a control antibody (open histograms). After incubated with secondary antibodies, the expression of the indicated BAFF receptors was analyzed by flow cytometry.³⁰  (B) Suppression of PKK expression inhibits I-κBα phosphorylation induced by BAFF. SUDHL-6 cells stably expressing shControl, shPKK-1, or shPKK-2 were treated with BAFF (200 ng/mL) for 15 minutes in serum-free medium. The expression levels of the indicated proteins were analyzed by Western blotting. The shRNA-expressing SUDHL-6 cells used here are the same cells described in Figure 1D. The PKK knockdown efficacy was confirmed to be at the levels shown in Figure 1D. (C) PKK knockdown leads to inhibition of nuclear translocation of NF-κB proteins. SUDHL-6 cells stably expressing the indicated shRNA were treated with BAFF (500 ng/mL) for 6 hours in serum-free medium. Nuclear and cytoplasmic fractions were prepared according to the instructions by the manufacturer (Active Motif), and the levels of the indicated NF-κB subunits in each fraction were analyzed by Western blotting. Analysis of MCM7 and γ-tubulin was used as the loading controls for nuclear and cytoplasmic fractions, respectively. (D) Suppression of PKK expression inhibits BAFF-induced NF-κB DNA-binding activity. SUDHL-6 cells expressing the indicated shRNA were treated as described in panel C, and the DNA-binding activity of the indicted NF-κB subunit was assayed as described in Figure 1C. The difference between control cells and cells expressing an shPKK in DNA-binding activity for all tested NF-κB subunits is significant (P < .01). (E) Effect of PKK knockdown on NF-κB promoter activation induced by BAFF and TNF-α. SUDHL-6 cells stably expressing the indicated shRNA were infected with a recombinant lentivirus carrying a firefly luciferase reporter regulated by 4-tandam copies of NF-κB consensus binding sequence (generously provided by John Ashton, University of Rochester). Forty hours after infection, the cells were treated with BAFF (500 ng/mL) and TFN-α (100 ng/mL), respectively, for the indicated times. The luciferase activity was measured as described previously.³⁶  Fold of activation was calculated by comparing the luciferase activity of BAFF or TNF-α stimulated cells with that of unstimulated cells. The means and standard deviations from 3 independent experiments are depicted. (F) Suppression of PKK expression inhibits BAFF-, but not TNF-α–, induced IκBα phosphorylation. SUDHL-6 cells stably expressing the indicated shRNA were treated with BAFF and TNF-α, respectively, for 3 minutes. The expression levels of the indicated proteins were analyzed by Western blotting. Analysis of IKKβ was used as a loading control. (G) SUDHL-6 cells expressing either shControl or shPKK-1 were treated with BAFF for the indicated time. IKK complex was immunoprecipitated with an IKKβ-specific antibody. The kinase activity of the immunoprecipitated IKK complex was assayed in vitro using GST-IκBα (amino acids 1-54) as a substrate (top). The amounts of IKKβ protein in the immunoprecipitated samples were analyzed by Western blotting using an IKKβ antibody (bottom).