Journal: bioRxiv
Article Title: Chemical Probe Discovery for DEAD-box RNA Binding Protein DDX21 using Small Molecule Microarrays
doi: 10.1101/2025.04.18.649545
Figure Lengend Snippet: KI-DX-014 blocks release of P-TEFb from 7SK snRNP and causes developmental defects in zebrafish via Pol II phosphorylation. a . Schematic of P-TEFb release assay in the presence of KI-DX-014. Created with BioRender.com . b . Western blot analysis of released P-TEFb from HEXIM1 immunopurified 7SK snRNP upon addition of purified DDX21-FL and ATP in the presence of KI-DX-014. c . Schematic of in vivo treatment in zebrafish embryo model. KI-DX-014 or vehicle (DMSO) were microinjected to fertilized zebrafish embryos at 1 cell stage. Reference CDK9 inhibitor (Flavopiridol) or vehicle (DMSO) containing medium were treated to fertilized zebrafish embryos at 1 cell stage. After 24 h, zebrafish embryos were dechorionated for imaging and total protein extraction. Created with BioRender.com . d , Representative images of zebrafish embryos at 24 h.p.f. treated with KI-DX-014 and reference CDK9 inhibitors; scale bar, 200 µm. e , Western blot analysis of phospho-RNA Pol II CTD serine 2 and serine 5 levels in zebrafish embryos after 24 h treatment with KI-DX-014 and reference CDK9 inhibitors.
Article Snippet: The antibodies used in this study include CDK9 (C12F7) Rabbit mAb (Cell Signaling, 2316, 1:1000), Anti-HEXIM1 antibody (Abcam, ab25388, 1:1000), Purified anti-RNA Polymerase II RPB1 Antibody (H5) (BioLegend, 920204, 1:1000), Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody (Abcam, ab5131, 1:1000), Goat anti-Mouse IgG (H+L) Poly-HRP Secondary Antibody, HRP (Invitrogen, 32230, 1:5000), Goat anti-Rabbit IgG (H+L) Poly-HRP Secondary Antibody, HRP (Invitrogen, 32260, 1:5000), Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, light chain specific (Jackson ImmunoResearch, 211-032-171, 1:5000)
Techniques: Phospho-proteomics, Release Assay, Western Blot, Purification, In Vivo, Imaging, Protein Extraction