Skip to main content
. 2023 Apr 3;12(4):1081–1093. doi: 10.1021/acssynbio.2c00587

Figure 5.

Figure 5

Homo- and heterobifunctional fratricide REULR. (A) TCGA cancer tissue RNA-seq data for RNF128, RNF130, RNF167, RNF43, and ZNRF3 was obtained from 17 cancer types, representing 21 cancer subtypes and were processed as median FPKM (number fragments per kilobase of exon per million reads) and visualized as a hierarchical clustering heatmap. (B) Schematic representation of homo- or heterobispecific fratricide REULR. (C) HEK293T cells were transiently transfected with HA-tagged full-length PA-TM-RING E3 ligase cDNA (human) under the control of a constitutively active CMV (cytomegalovirus) promoter, as indicated. 24 h post-transfection, cells were incubated with fratricide REULR molecules (50 nM), targeting RNF128, RNF130, RNF167, RNF43, or ZNRF3. (D) HEK293T cells were co-transfected with HA-tagged full-length RNF43 and MYC-tagged full-length ZNRF3 cDNA (human) and treated with a heterobispecific RNF43-ZNRF3 fratricide REULR 24 h post-transfection (monomeric binding moieties were used as negative controls). After 24 h, cells were subjected to FACS analysis using a HA antibody (Alexa Fluor 647 conjugate) or an MYC antibody (Alexa Fluor 488 conjugate) to monitor PA-TM-RING E3 ligase levels on the cell surface. Data are mean ± s.d. (n = three replicates). (E) Schematic representation of heterobispecific arrayed multimeric fratricide REULR. (F) HEK293T cells were co-transfected with HA-tagged full-length RNF128, MYC-tagged full-length ZNRF3, and FLAG-tagged full-length RNF43 cDNA (human). 24 h post-transfection, cells were treated with homo- or heterobispecific Fratricide REULR molecules as indicated or a RNF43-RNF128-ZNRF3 multimeric fratricide REULR (PBS and monomeric binding moieties were used as negative controls). After 36 h, cells were subjected to FACS analysis using an HA antibody (Alexa Fluor 647 conjugate), a MYC antibody (Alexa Fluor 488 conjugate), and a FLAG antibody (Brilliant Violet 421) to monitor RNF128 (left panel), RNF43 (middle), and ZNRF3 (right panel) E3 ligase levels on the cell surface. Data are mean ± s.d. (n = three replicates).