Fig. 6

NF-κB activity is important for protein stability. a, b Jurkat T-cells were transfected with EF1-α-driven Tax expression plasmids Tax (wildtype) or Tax mutant M22 (NF-κB-deficient; 40 µg each), Tax together with pIκBα-DN (10 µg), a dominant negative inhibitor of κB (IκBα-DN), or M22 together with pc-FLAG-IKK2-EE (40 µg), a constitutive active variant of IKK2 (IKK2-EE). All samples were replenished to 100 µg DNA with the empty vector pcDNA. At 24 h after transfection, cells were treated for the indicated periods of time with 50 µg/ml cycloheximide (CHX). a Immunoblot shows Tax and ACTB. Numbers indicate representative densitometric analysis of Tax protein expression levels normalized on Tax expression at 0 h CHX. b Densitometric values derived from (a), relative to 0 h CHX, are depicted for Tax (closed squares), Tax together with IκBα-DN (closed diamonds), M22 (closed triangles) and M22 together with IKK2-EE (black crosses). Each experiment was independently performed in quadruplicate and values ± SE are shown. Protein half-life of Tax or M22, respectively, were empirically determined as indicated. c, d Jurkat T-cells were transfected with Tax-1 expression plasmids Tax (wildtype; 40 µg), M22 (NF-κB-deficient) or M7 (CREB- and NF-κB-deficient; 100 µg each), all in the pEF-1α backbone, replenished, where necessary, to 100 µg DNA with empty vector DNA. At 24 h after transfection, transfected cells were treated, where indicated, for 24 h with the water-dissolved lysosome inhibitor Ammonium chloride (NH₄Cl, 20 mM), the ubiquitin-activating enzyme E1 inhibitor 4[4-nitro-furan-2-ylmethylene)-3,5-dioxo-pyrazolidin-1-yl]-benzoic acid ethyl ester (PYR-41; 15 µM) or the solvent control DMSO. c Western Blot shows Tax and GAPDH. Numbers indicate a representative densitometric analysis of Tax protein expression levels normalized on GAPDH and the respective Tax expression in mock or DMSO treated cells. d Densitometric analysis of Tax protein, normalized on GAPDH and the respective Tax expression in mock or DMSO treated cells, of four independent experiments was performed. Values ± SE are depicted and were compared using student’s t-test (*p < 0.05; **p < 0.01). e Schematic working model of a potential positive feedback loop between Tax and NF-κB activity. Components of the NF-κB signaling cascade with an experimentally shown impact on Tax protein expression are colored in red