Figure 4
A central region in RNF169 and a conserved region in the N-terminus of DYRK1A are necessary for the DYRK1A-RNF169 interaction. (A,B) Interaction experiments using HEK-293T cells transiently transfected with the RNF169 and DYRK1A expression constructs indicated, and immunoprecipitated with anti-HA (A) or anti-FLAG (B) Abs. Schematic illustrations of the RNF169 (A) or DYRK1A (B) primary structures and the deletion mutants used: HIS, histidine tract; KD, kinase ___domain; LR, leucine-arginine ___domain; MIU, motif interacting with ubiquitin; NLS, nuclear localization signal; PEST, PEST region; RING, Really Interesting New Gene ___domain; S/T, region rich in serine and threonine. (C) Soluble extracts from HEK-293T cells transiently expressing the proteins indicated were used in anti-FLAG co-immunoprecipitation experiments (see Fig. S4D for the sequence of the deletions). (D) Soluble extracts from HEK-293T cells expressing either aa 79–113 of DYRK1A fused to GST-GFP or GST-GFP alone were subjected to affinity purification using glutathione-Sepharose beads. Both the lysates and the GST-bound fractions were analyzed in WBs probed with Abs to the proteins indicated, and to GFP to detect the fusion proteins: *non-specific band. (E) WBs of anti-FLAG immunoprecipitates of HEK-293T soluble extracts expressing FLAG-RNF169. (F) Pull-down experiment with MBP-RNF169 expressed in bacteria as bait. HEK-293T extracts expressing FLAG-DYRK1A alone or together with HA-DCAF7 were used as preys, and both the lysates and the bound proteins were analyzed in WBs probed with antibodies to the tags.
