Figure 3.
Non-canonical NF-κB activity in an ectopic expression system and kinase assays. (A) Schematic description of the experiment. RELA−/− HEK293T cells were transfected with an NF-κB firefly luciferase reporter and thymidine-kinase Renilla luciferase reporter plasmid together with RELB and NFKB2 coding plasmids. Additional transfection of either an EV plasmid or containing the coding sequence of wt IKKα or mutants IKKα allowed the assessment of their own activity toward the activation of the non-canonical NF-κB pathway. (B) Relative luciferase activity resulting from the transfection of an increasing dose of a plasmid coding IKKα wt, p.H142R, p.P190L, p.Y580C, p.Q422*, or p.K44A (from 20 to 70 ng as indicated), co-transfected with RELB and p100. The NF-κB Firefly signal was normalized to the Renilla signal and then to transfection with RELB p100 only. An EV was co-transfected in each condition to reach a constant amount of plasmid. Statistical analysis was performed using a One-Way ANOVA with multiple comparisons by Dunn’s test (****P < 0.0001, ***P < 0.001). Representative of at least three experiments. (C) Western blot of phosphorylated IKKα/β before (whole cell lysate/input) and after immunoprecipitation of HA-tag IKKα transfected in HEK293T cells (IP HA-Tag). Representative of three experiments. (D) IKKα kinase activity after pulldown of the different alleles expressed as the relative luminescence of the wt allele or the variants to the EV. Each point represents one experiment. Statistical analysis was performed using a one-Way ANOVA with multiple comparisons by Dunn’s test (*P < 0.05). (E) Western blot showing the phosphorylated form (S32) of a GST-IκΒα protein after incubation with the pulled-down IKKα (or EV). All figures are representative of at least two independent experiments. Error bars represent mean ± SD in all barplots. Source data are available for this figure: SourceData F3.

Non-canonical NF-κB activity in an ectopic expression system and kinase assays. (A) Schematic description of the experiment. RELA−/− HEK293T cells were transfected with an NF-κB firefly luciferase reporter and thymidine-kinase Renilla luciferase reporter plasmid together with RELB and NFKB2 coding plasmids. Additional transfection of either an EV plasmid or containing the coding sequence of wt IKKα or mutants IKKα allowed the assessment of their own activity toward the activation of the non-canonical NF-κB pathway. (B) Relative luciferase activity resulting from the transfection of an increasing dose of a plasmid coding IKKα wt, p.H142R, p.P190L, p.Y580C, p.Q422*, or p.K44A (from 20 to 70 ng as indicated), co-transfected with RELB and p100. The NF-κB Firefly signal was normalized to the Renilla signal and then to transfection with RELB p100 only. An EV was co-transfected in each condition to reach a constant amount of plasmid. Statistical analysis was performed using a One-Way ANOVA with multiple comparisons by Dunn’s test (****P < 0.0001, ***P < 0.001). Representative of at least three experiments. (C) Western blot of phosphorylated IKKα/β before (whole cell lysate/input) and after immunoprecipitation of HA-tag IKKα transfected in HEK293T cells (IP HA-Tag). Representative of three experiments. (D) IKKα kinase activity after pulldown of the different alleles expressed as the relative luminescence of the wt allele or the variants to the EV. Each point represents one experiment. Statistical analysis was performed using a one-Way ANOVA with multiple comparisons by Dunn’s test (*P < 0.05). (E) Western blot showing the phosphorylated form (S32) of a GST-IκΒα protein after incubation with the pulled-down IKKα (or EV). All figures are representative of at least two independent experiments. Error bars represent mean ± SD in all barplots. Source data are available for this figure: SourceData F3.

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