Fig. 4

RSK3 controls proteasome activity and IκBα degradation. A, B HMECT were infected with pWZL/RSK3 (RSK3) or control pWZL vector (Ctrl), selected for 1 week with neomycin and plated for indicated assays. A Western blot characterization of pIκBα and IκBα levels. Cells were stimulated with TNFα for 5 min. B Quantification of the Western blots of panel A, normalization with tubulin (n = 4, ratio paired t-test was used to determine statistical significance). C, D 293GP cells were transfected with pFLAG-CMV2 (Ctrl) or pFLAG-CMV2/RSK3 (RSK3) and 48 h after collected for FLAG immunoprecipitation. C Silver staining of FLAG-immunoprecipitated proteins before mass spectromety analysis. D FLAG-immunoprecipitation and Western blot against the indicated tag and proteins (representative of 3 independent experiments). E Proteasome enrichment. Assay was performed in HMECT with Cyclex Proteasome enrichment kit and a further Western blot was performed (representative of 3 independent experiments) Ctrl = control resin, UBL = UBL resin, binding to the proteasome. F Proteasome activity assay performed in HMECT constitutively expressing or not RSK3 (n = 4 independent experiment, ratio-paired t-test was used to determine statistical significance). G RT-QPCR of NF-κB-dependent SASP encoding mRNA in HMECT expressing or not RSK3 and RSK3 mutants (n = 3 independent experiments). Ratio-paired t-test was used to determine statistical significance