Journal: Nature

Article Title: Antibody targeting of E3 ubiquitin ligases for receptor degradation.

doi: 10.1038/s41586-022-05235-6

Figure Lengend Snippet: Fig. 1 | The Wnt-responsive E3 ubiquitin ligases RNF43 and ZNRF3 can degrade IGF1R. a, Left, CRISPR–Cas9 strategy used to generate APC mutant organoids. Right, phase-contrast images of wild-type (WT) and APC truncation mutant (Apc−/−) colon organoids. Data are representative of two independent experiments. Scale bars, 250 μm. b, Gene expression analysis of wild-type and Apc−/− colon organoids. Genes encoding proteins with predicted transmembrane domains (TM) (grey), E3 ubiquitin (Ub) ligases (light blue) or both (dark blue) are shown. Data are the mean expression from three independent experiments. FC, fold change; FDR, false discovery rate. c, In situ hybridization in AKPS colon organoids probing for Rnf43 and Znrf3. Data are representative of two independent experiments. Scale bars, 50 μm. d, Schematic representation of the iDimerize construct. HA, haemagglutinin tag. e, Levels of total and immunoprecipitated IGF1R and RNF43 following HA tag immunoprecipitation (IP) following treatment of HEK293T cells harbouring the RNF43–IGF1R iDimerize construct

Article Snippet: Cleared lysates were incubated with 25 μl per sample prewashed Dynabeads protein G (Thermo Fisher, 10004D) conjugated with IGF1 receptor (D23H3) XP rabbit monoclonal antibody (Cell Signaling; 9750) for IGF1R immunoprecipitation at 1.87 μg per sample.

Techniques: Ubiquitin Proteomics, CRISPR, Mutagenesis, Gene Expression, Expressing, In Situ Hybridization, Construct, Immunoprecipitation

Journal: Nature

Article Title: Antibody targeting of E3 ubiquitin ligases for receptor degradation.

doi: 10.1038/s41586-022-05235-6

Figure Lengend Snippet: Fig. 2 | Tethering endogenous RNF43 or ZNRF3 to IGF1R induces target internalization and degradation. a, Schematic representation of ligase- based PROTAB-mediated IGF1R degradation.b,c, Levels of cell-surface IGF1R assessed by flow cytometry following treatment of HT29 cells expressing gD–RNF43–Flag (b) or gD–ZNRF3–Flag (c) with campaign PROTABs for 24 h. Graphs depict IGF1R clearance versus bivalent affinities of ligase campaign antibodies from one high-throughput FACS screening campaign. d, Levels of cell-surface IGF1R following treatment of HT29 HiBiT-IGF1R knock-in (KI) cells with ZNRF3*IGF1R bispecific PROTABs for 24 h. The graph depicts IGF1R clearance from one screening campaign with values from technical repeats overlayed. Assay controls and PROTABs used subsequently are highlighted. e, Levels of IGF1R-β following treatment of various CRC cell lines with indicated antibodies for 24 h. Per cent degradation is normalized to Cixu*NIST treatment and is the average of two independent experiments. Expression levels of key

Article Snippet: Cleared lysates were incubated with 25 μl per sample prewashed Dynabeads protein G (Thermo Fisher, 10004D) conjugated with IGF1 receptor (D23H3) XP rabbit monoclonal antibody (Cell Signaling; 9750) for IGF1R immunoprecipitation at 1.87 μg per sample.

Techniques: Flow Cytometry, Expressing, High Throughput Screening Assay, Knock-In

Journal: Nature

Article Title: Antibody targeting of E3 ubiquitin ligases for receptor degradation.

doi: 10.1038/s41586-022-05235-6

Figure Lengend Snippet: Fig. 3 | ZNRF3*IGF1R bispecific PROTABs induce target degradation in a ligase-dependent manner. a, Clearance of IGF1R in cells treated with PROTABs (Fig. 2e) versus the basal IGF1R:ZNRF3 cell-surface ratio determined by copy number analysis (Extended Data Fig. 3a,b). b, Levels of cell-surface ZNRF3 in indicated cell lines. Data are representative of two independent experiments. c, Levels of IGF1Rβ in HT29 cells endogenously expressing wild-type or indicated ZNRF3 indels following treatment with 10 μg ml−1of the indicated PROTABs for 24 h. β-Tubulin was used as a loading control. Data are representative of two independent experiments. d, Levels of IGF1Rβ and ubiquitin in SW48 cells treated with DMSO or E1 inhibitor MLN7243 for 2 h followed by PROTAB treatment for 6 h. Data are representative of three independent experiments. e, Levels of total and immunoprecipitated IGF1Rβ and ubiquitin in HT29 cells subjected to denaturing IGF1Rβ immunoprecipitation (IP) following treatment

Article Snippet: Cleared lysates were incubated with 25 μl per sample prewashed Dynabeads protein G (Thermo Fisher, 10004D) conjugated with IGF1 receptor (D23H3) XP rabbit monoclonal antibody (Cell Signaling; 9750) for IGF1R immunoprecipitation at 1.87 μg per sample.

Techniques: Expressing, Control, Ubiquitin Proteomics, Immunoprecipitation

Journal: Nature

Article Title: Antibody targeting of E3 ubiquitin ligases for receptor degradation.

doi: 10.1038/s41586-022-05235-6

Figure Lengend Snippet: Fig. 4 | Multiple cell-surface E3 ubiquitin ligases can degrade plasma membrane targets. a,b, Levels of HER2 (a) and PD-L1 (b) in SW48 xenografts 48 h after intraperitoneal administration with indicated antibodies. GAPDH was used as a loading control. n = 4 mice per group. c, Frequency of detectable cell-surface gD epitopes in HT29 cells expressing indicated gD–ligase–Flag constructs versus parental cells. Graph depicts gD-positive cells from three independent experiments. Data are mean ± s.e.m. with values from biological repeats overlaid. d, Levels of IGF1Rβ in HT29 cells expressing indicated gD– ligase–Flag constructs following treatment with specified antibodies for 24 h. Data are representative of three independent experiments. e, Levels of cell-surface FZD5 in ASPC1 cells expressing indicated gD–ligase–Flag constructs following antibody treatment at 1 or 10 µg ml−1 for 24 h. Data are presented as lines connecting mean values from two independent experiments with values from biological repeats overlaid. f, Schematic representation of

Article Snippet: Cleared lysates were incubated with 25 μl per sample prewashed Dynabeads protein G (Thermo Fisher, 10004D) conjugated with IGF1 receptor (D23H3) XP rabbit monoclonal antibody (Cell Signaling; 9750) for IGF1R immunoprecipitation at 1.87 μg per sample.

Techniques: Ubiquitin Proteomics, Clinical Proteomics, Membrane, Control, Expressing, Construct