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: # Name Contents Resistance Marker Backbone Addgene Used for 1 Artichoke SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR AmpicillinR Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) #73320 Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen 2 Cilantro 2 PGK.BsmBICloneSite- 10aaFlexibleLinker -EGFP.IRES.mCherry. cppt.EF1α.PuroR #74450 C2H2 zinc finger library screen, C2H2 zinc fingers in isolation Open in a separate window Degradation Reporter Vectors

Techniques: Titration, Construct, Plasmid Preparation, Expressing, Flow Cytometry, Fluorescence, Sequencing, 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: # Name Contents Resistance Marker Backbone Addgene Used for 1 Artichoke SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR AmpicillinR Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) #73320 Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen 2 Cilantro 2 PGK.BsmBICloneSite- 10aaFlexibleLinker -EGFP.IRES.mCherry. cppt.EF1α.PuroR #74450 C2H2 zinc finger library screen, C2H2 zinc fingers in isolation Open in a separate window Degradation Reporter Vectors

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: # Name Contents Resistance Marker Backbone Addgene Used for 1 Artichoke SFFV.BsmBICloneSite- 17aaRigidLinker -EGFP.IRES.mCherry.cppt.EF1α.PuroR AmpicillinR Lentiviral (pLKO, taken from lentiGuide-Puro, Addgene #52963) #73320 Full-length proteins, IKZF3 aa130–189, saturation mutagenesis screen 2 Cilantro 2 PGK.BsmBICloneSite- 10aaFlexibleLinker -EGFP.IRES.mCherry. cppt.EF1α.PuroR #74450 C2H2 zinc finger library screen, C2H2 zinc fingers in isolation Open in a separate window Degradation Reporter Vectors

Techniques: Marker, Mutagenesis, Zinc-Fingers, Isolation

Journal: Journal of experimental botany

Article Title: Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses.

doi: 10.1093/jxb/erae278

Figure Lengend Snippet: Fig. 1. Three-dimensional structure of a typical C2H2-type zinc finger protein. (A) Protein sequence alignment of the 3-D structure composed of H1–H6 helix strands and beta sheets. (B) Normal representation of the 3-D structure showing five Zn metal ions. (C) Example of C2H2-type zinc finger proteins interacting with Zn metal ions. The amino acids on (C) are the catalytic sites (two HIS507, HIS511 and CYS491, CYS4940) of the protein.

Article Snippet: Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

Techniques: Sequencing

Journal: Journal of experimental botany

Article Title: Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses.

doi: 10.1093/jxb/erae278

Figure Lengend Snippet: Fig. 2. Classification of C2H2-type zinc finger proteins into subgroups in Arabidopsis. The image was created using Figdraw (https://www.figdraw. com).

Article Snippet: Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

Techniques:

Journal: Journal of experimental botany

Article Title: Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses.

doi: 10.1093/jxb/erae278

Figure Lengend Snippet: Fig. 3. The regulatory network of C2H2-type zinc finger proteins in responses to abiotic stresses. ABA, abscisic acid; APX, ascorbate peroxidase; AsA, ascorbic acid; CAT, catalase; GA, gibberellin; JA, jasmonic acid; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; POD, peroxidase; ROS, reactive oxygen species; SA, salicylic acid; SOD, superoxide dismutase.

Article Snippet: Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

Techniques:

Journal: Journal of experimental botany

Article Title: Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses.

doi: 10.1093/jxb/erae278

Figure Lengend Snippet: Fig. 4. The regulatory network of C2H2 zinc finger proteins in response to biotic stress. ABA, abscisic acid; ETI, effector-triggered immunity; H2O2, hydrogen peroxide; HR, hypersensitive response; JA, jasmonic acid; PTI, (pathogen-associated molecular pattern-triggered immunity; SA, salicylic acid.

Article Snippet: Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

Techniques:

Journal: Journal of experimental botany

Article Title: Recent advances in the multifaceted functions of Cys2/His2-type zinc finger proteins in plant growth, development, and stress responses.

doi: 10.1093/jxb/erae278

Figure Lengend Snippet: Fig. 5. The regulatory network of C2H2 zinc finger proteins in response to plant growth and development. ABA, abscisic acid; CK, cytokinin; GA, gibberellin; JA, jasmonic acid.

Article Snippet: Genome-wide study of C2H2 zinc finger gene family in Medicago truncatula.

Techniques:

Journal: bioRxiv

Article Title: The structural basis of Indisulam-mediated recruitment of RBM39 to the DCAF15-DDB1-DDA1 E3 ligase complex

doi: 10.1101/737510

Figure Lengend Snippet: a , Bioinformatics workflow for identifying proteins bearing putative degron motif required for DCAF15-Indisulam recruitment (left) and structures of DCAF15-bound Indisulam (orange) and RBM39(RRM2) (magenta) with central alpha helix highlighted (right). The RBM39 residues found to be most critical for DCAF15-Indisulam recruitment are labeled (M265, G268, E271, and P272). 6,475 proteins with known structures were identified in the Swiss-Prot database, of which 3,425 had a glycine in an alpha helix. 3,112 of the glycine-containing alpha helices aligned to an RBM39(RRM2) structure (2JRS) with RMSD <2.0 Å. Among these matches, only RBM23 and RBM39 helices had a sequence matching the required X 1 XXM 4 XXG 7 XXEP motif. RMSD values, PDB IDs, and gene names shown in bottom table. b , Volcano plot summary of expression proteomics experiments comparing lysates from HCT-116 cells treated for 4 h with 10 µM Indisulam or DMSO. Significant downregulated proteins (p value < 1E-2, Log 2 fold-change) are labeled. Data represents two (n=2) biological replicates per treatment condition in a single experiment. c , Western blots showing levels of RBM39, RBM23, and ZNF277 in HCT116 cells following 4 h treatment with varied concentrations of Indisulam or DMSO. Data shown from one individual, representative experiment from three independent repeats.

Article Snippet: Blots were incubated with primary antibody solutions made in TBS-T with 5% milk for RBM39 (Sigma, Cat #HPA001591, 1:2500) GAPDH (CST, Cat #2118L, 1:1000), Vinculin (Cell Signaling Technology, Cat# 13901S, 1:1000), ZNF277 (Pro-Sci, Cat #46-616, 1:1000), RBM23 (Invitrogen, Cat# PA5-52060, 1:1000), or DDA1 (Proteintech, Cat #14995-1-AP, 1:1000) overnight at 4°C.

Techniques: Labeling, Sequencing, Expressing, Western Blot