Extended Data Figure 9. RNF146 directly interacts with TNKS.

a, (Left) SEC profiles of untagged TNKS(5ARC) (blue), His₆T7-RNF146 (red), and a 1:1 mixture of these proteins (green). Numbers above the peaks indicate the average mass obtained by multi-angle static light scattering (MALS) for each peak. His₆T7-RNF146 and TNKS(5ARC) co-migrate as a single peak with an apparent mass of 128 kDa. (Right) Coomassie-stained SDS-PAGE analysis of the SEC peaks in left panel show the presence of both proteins within the peak of the TNKS(5ARC)/His₆T7-RNF146 complex (Bottom right). b, GST pull-down of partially purified full-length mouse tankyrase-1 (FL-mTNKS1) with GST tagged RNF146-R163A (PAR-binding deficient RNF146 mutant)¹¹. Full-length mTNKS1 can be pulled down by GST-RNF146, but not GST. c, Co-immunoprecipitation of HA-RNF146 variants with transiently transfected flag-tagged TNKS-M1207V (catalytically inactive mutant). The M1207V mutation prevents auto-PARylation of TNKS and therefore PAR-mediated interactions between RNF146 and TNKS³⁸. Under the experimental conditions, both the motif I mutant, G199V, and the motifs I+IV mutant, G199V/G337V/G338V, significantly reduce the RNF146-TNKS interaction. d, Coomassie-stained SDS-PAGE of proteins used in the GST pull-down assay shown in Figure 4a (inputs). Samples were used in a 1:2 ratio (3 μM GST-RNF146 to 6.7 μM TNKS(5ARC)) for these GST pull-down experiments.