Long-range enhancer-controlled genes are hypersensitive to regulatory factor perturbations Tjalsma et al.
Description
To study the contribution of regulatory factors to long-range enhancer-mediated gene regulation, we created degron cell lines for the factors RAD21, LDB1, and MED23, in human K562 cells. By endogenously tagging these proteins with a degron tag, we were able to quickly deplete them and measure their effect on gene expression using nascent RNA-sequencing. These files contain raw western blot images used to confirm that treatment of these cells with the degron ligand resulted in depletion of the tagged factors.
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Steps to reproduce
Human erythroleukemia K562 cells (female) were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich) and 1% pen–strep (Gibco). Cells were grown at a maximal density of 5x10^5 cells/ml. RAD21-AID, LDB1-FKBP or MED23-FKBP K562 degron cells were treated with 5-Ph-IAA (RAD21), or dTAG7 (LDB1/MED23), or DMSO as control. After 4 hours, equal amounts of cells were pelleted and directly resuspended in Laemmli buffer. After boiling at 95 degrees for 10 minutes, samples were loaded on a 4–15% Mini-PROTEAN TGX Precast Protein Gel (Biorad Cat#4561083). Proteins were transferred to a PVDF membrane, which was blocked with 5% milk in PBS-T (0.1% Tween-20 in PBS), and incubated overnight at 4 degrees with indicated antibodies. Antibodies used were: FLAG (F1804, Merck, 1:1000), RAD21 (05-908, Merck, 1:1000), GAPDH (sc-32233, Santa Cruz, 1:1000), SF3B1 (sc-514655, Santa Cruz, 1:100). After washing with PBS-T, secondary antibody anti-mouse (W4021, Promega, 1:5000) was added to the blot for 1 hour at room temperature. Blots were developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (ThermoFisher) and visualized with a ImageQuant 800 imager (Amersham).