Fig. 4: Mutagenesis analysis of Pirh2 for Ala6/C-degron binding.
From: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2
a ITC fitting curves of wild-type (WT) and mutant Pirh2 titrated with the Ala6/C-degron peptide. The corresponding mutant proteins and binding affinities (KD) are indicated. NB, no detectable binding under our experimental conditions. b Schematic representation of the GPS assay. PCMV, cytomegalovirus promoter; DsRed, Discosoma sp. red fluorescent protein; IERS, internal ribosome entry site; GFP, green fluorescent protein. c Stability analysis of GFP-fused Ala6/C-degron in endogenous Pirh2 knockdown cells, and rescued by exogenously expressed WT and mutant Pirh2. The shRNA targets the 3′-untranslated region (UTR) of endogenous Pirh2. The ratio of GFP/DsRed was analyzed by flow cytometry. d Western blot analysis of HA-tagged WT and mutant Pirh2 expression in Ala6/C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. 9.
