Figure 2
From: SQSTM-1/p62 potentiates HTLV-1 Tax-mediated NF-κB activation through its ubiquitin binding function
p62 potentiates Tax-dependent NF-κB activation upstream of IKK activation. (a) Wild type (WT) and p62−/− MEF cells were transfected with Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. (b) WT and p62−/− MEF cells were transfected with Tax-His. After fractionation of cell nuclei, transcriptionally active p65 was quantified by ELISA. (c) Lysates from WT and p62−/− MEF cells transfected with Tax-His were analyzed by western blot. (d) WT and p62−/− MEF cells were transfected with Tax-His. After RNA extraction and conversion to cDNAs, Il6 and gapdh cDNAs were amplified by PCR (left panel). The normalized Il6 signal intensities were calculated and are shown relative to the corresponding Tax-negative conditions on the graph (results from 2 independent experiments). Cell lysates were also analyzed by western blot (right panel). (e) HEK293T cells were transfected with control (siCTRL) or p62-specific (sip62) siRNA and Tax-His, together with an NF-κB-luc construct. Luciferase activity was measured and normalized over the corresponding Tax-negative condition. The graph shows results from at least 3 independent experiments. (f) Lysates from HEK293T cells transfected with siCTRL or sip62 and Tax-His were analyzed by western blot. (g) Jurkat cells were transfected with increasing amounts of Myc-p62 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 endogenous p62 were set to 1 and other values are shown as fold change over the “endogenous p62” condition. The graph shows results from 3 independent experiments. (h) Lysates from Jurkat cells were analyzed by western blot. ***p < 0.001; **p < 0.01; *p < 0.05 (one-way ANOVA with Bonferroni post-hoc test). Full-length blots and gels are presented in Supplementary Fig. S4.
