Ribosomal protein S3 interacts with the NF-κB inhibitor IκBα

•RPS3 interacts with the NF-κB inhibitor IκBα in resting cells and in vitro.•The ankyrin-repeats of IκBα mediate the interaction with the RPS3 C-domain.•IκBα facilitates the formation of a cytoplasmic complex with RPS3 and p65. Ribosomal protein S3 (RPS3) is part of nuclear, transcriptionally active and cytoplasmic inhibitory complexes containing NF-κB variant p65. We show that in resting HEK293 cells, RPS3 interacts with NF-κB inhibitor IκBα. In contrast, efficient co-precipitation of p65 with RPS3 was only achieved in the presence of ectopic IκBα. In addition, a strong in vitro interaction was observed between RPS3 and IκBα, while binding between RPS3 and p65 was very weak. Furthermore, IκBα facilitated the reconstitution of p65 and RPS3 into one complex in vitro. Our results suggest that IκBα sequesters not only p65 but also RPS3 in the cytoplasm. This would ensure maintenance of an RPS3 pool for the NF-κB pathway as well as equimolar release of RPS3 and p65 upon stimulation. RPS3physically interacts with p65 and I kappa B-alpha by anti tag coimmunoprecipitation (View interaction) RPS3 and I kappa B-alphaphysically interact with p65 by pull down (View interaction) I kappa B-alphaphysically interacts with RPS3 by anti tag coimmunoprecipitation (1, 2, 3)