Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: (A) TR-FRET counter titration. Unlabeled IKZF1 constructs (0.01–100 μM) titrated to preassembled Alexa488DDB1ΔB-CRBN-pomalidomide-biotinIKZF1ZF1−2−3 (200 nM Alexa488DDB1ΔB-CRBN, 100nM biotinIKZF1ZF1−2−3, 5μM pomalidomide). (B) Schematic of the protein degradation reporter vector (IRES: Internal ribosome entry site). (C) HEK293T WT and CRBN−/− cells expressing IKZF3 constructs in the degradation reporter were treated for 20 hours with DMSO or drug then analyzed by flow cytometry to quantify the DMSO-normalized ratio of eGFP/mCherry fluorescence (exp. rep. = 3, tech. rep. = 3, bar heights indicate mean of experimental replicates, error bars indicate 95% CI). (D) Schematic of the human C2H2 ZF library screen. (E) Average fold-depletion of sequencing read counts (DMSO/drug) and corresponding p values (empirical rank-sum test-statistic) for the 5,611 C2H2 ZFs with raw read count >200 in all three control replicates (exp. rep. = 3, labeled data points possess FDR<0.01 in at least one of the three drugs).

Article Snippet: “Cilantro 2”, which possesses a 10aa flexible linker at the N-terminus of eGFP, was used for individual C2H2 zinc fingers (Addgene #74450, Benchling link to vector map and sequence: https://benchling.com/s/k2PYU86Q ).

Techniques: Titration, Construct, Plasmid Preparation, Expressing, Flow Cytometry, Fluorescence, Sequencing, Control, Labeling

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: (A) Sequence alignment of the 11 C2H2 ZFs with FDR<0.01 in at least one drug condition (amino acids colored by property). (B) Saturation mutagenesis screen of IKZF3 aa 130–189 in presence of lenalidomide displayed as heat map of the FDR for mutant amino acids (unpaired, one-sided t-test, FDR correction performed within each column, tech. rep. = 3). Asterisks indicate amino acids required for the ZF fold and arrows indicate non-structural IKZF3 residues required for degradation. Complete results for all 60 amino acids are located in fig. S3C (C) ANOVA p values for difference in frequency of mutant amino acids at each position in IKZF3 aa 130–189 (DMSO vs drug). Complete results for all 60 aa are located in fig. S3D. (D) HEK293T cells expressing IKZF3 ZF2 constructs in the degradation reporter were treated for 20 hours with DMSO or 1μM drug after which flow cytometry was used to measure the DMSO-normalized ratio of eGFP/mCherry fluorescence (exp. rep. = 1, tech. rep. = 3, bar height is the average of tech. rep, error bars denote 95% CI).

Article Snippet: “Cilantro 2”, which possesses a 10aa flexible linker at the N-terminus of eGFP, was used for individual C2H2 zinc fingers (Addgene #74450, Benchling link to vector map and sequence: https://benchling.com/s/k2PYU86Q ).

Techniques: Sequencing, Mutagenesis, Expressing, Construct, Flow Cytometry, Fluorescence

Journal: Science (New York, N.Y.)

Article Title: Defining the human C2H2 zinc-finger degrome targeted by thalidomide analogs through CRBN

doi: 10.1126/science.aat0572

Figure Lengend Snippet: Degradation Reporter Vectors

Article Snippet: “Cilantro 2”, which possesses a 10aa flexible linker at the N-terminus of eGFP, was used for individual C2H2 zinc fingers (Addgene #74450, Benchling link to vector map and sequence: https://benchling.com/s/k2PYU86Q ).

Techniques: Marker, Mutagenesis, Zinc-Fingers, Isolation