Figure 6
From: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function
p62 binding to ubiquitin is required for p62 potentiation of Tax-mediated NF-κB activation. (a) Jurkat cells were transfected with full-length Myc-p62 (My-p62 FL) or p62 mutants in which the Tax-interacting region (Myc-p62 Δ170-221) or the ubiquitin-binding domain (Myc-p62 ΔUBA) were deleted, and an NF-κB-luc construct, followed by transduction with an empty or Flag-Tax-encoding lentivector. Luciferase activity was measured and normalized to the corresponding Tax-negative condition. Values obtained with full-length ectopic p62 were set to 1 and other values are shown as fold change over the “p62 FL” condition. The graph shows results from 3 independent experiments. (b) Lysates from p62−/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 Δ170-221 were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. (c) Lysates from p62−/− MEF cells transiently expressing Tax-His and either Myc-tagged full-length p62 (p62FL) or p62 ΔUBA were immunoprecipitated with a Myc-specific antibody followed by western blot analyses. (d) HeLa cells were transfected with control (−) or p62-specific (+) siRNA and Tax-His. Cell lysates were submitted to a His-specific Ni-NTA pulldown in denaturing conditions before western blot analyses. (e) Lysates from WT and p62−/− MEF cells transiently expressing Tax-His- and FLAG-IKKγ were immunoprecipitated with a FLAG-specific antibody followed by western blot analyses. **p < 0.01; ns, p > 0.05 (one-way ANOVA with Bonferroni post-hoc test). Full-length blots are presented in Supplementary Fig. S4.
