Fig. 2: Validating E3IDP45 for the TCD system.
See also Supplementary Figs. 3, 4. a The schematic of the pilot assays involving transient expression of the X–E3 degrader with the X–YFP target in N. benthamiana or protoplasts to assess TCD efficiency via microscopic observation or immunoblot analysis. X–YFP, YFP-tagged target protein X. X–E3, the degrader for X after fusing with E3. b, c Degradation of the E3IDP45ΔRING–YFP by the E3IDP45 degrader in the pilot experiments of microscopic observation (b) and immunoblot analysis (c) in N.benthamiana. d Microscopic observation of each X–YFP after co-expression with different X–E3IDP45 degraders. Six transcription factors and the E3IDP45ΔRING were examined as the target X protein. e, f Immunoblot analysis of E3IDP45ΔRING–YFP (e) and PLP308–YFP (f) after co-expression with different X–E3IDP45 degraders. g Co-immunoprecipitation to show the interaction between E3IDP45ΔRING with the LCD domain deleted (E3IDP45ΔRING, ΔLCD) and E3IDP45ΔRING. h, i Degradation of E3IDP45ΔRING–YFP without the LCD in the target (E3IDP45ΔRING, ΔLCD) by the E3IDP45 degrader in the pilot experiments of microscopic observation (h) and immunoblot analysis (i) in N.benthamiana. j In vitro ubiquitination assay to evaluate the self-ubiquitination capacity of E3IDP45ΔLCD. k, l Degradation of the E3IDP45ΔRING–YFP target by degraders of E3IDP45 without the LCD in the degrader (E3IDP45ΔLCD) in the pilot experiments of microscopic observation (k) and immunoblot analysis (l) in N.benthamiana. Coomassie brilliant blue (CBB; c) or Ponceaus S staining (e, f, i, l) was used as a control for protein loading. Semi-quantitative RT-PCR was conducted against YFP with NbUBQ as the internal control (c, e, f, i, l). For microscopic observation, 35S::CFP serves as the control (b, h, k). Scale bar, 10 µm (b, d, h, k). Source data are provided as a Source Data file.
