Journal: Science Advances
Article Title: Nuclear accumulation of YTHDF1 regulates mRNA splicing in the DNA damage response
doi: 10.1126/sciadv.ado7660
Figure Lengend Snippet: ( A ) Immunofluorescence detection of YTHDF1 using anti-YTHDF1 in HEK293T cells with or without x-ray treatment (a dose of 4 Gy). Nuclei were counterstained with Hoechst (blue). Scale bars, 10 μm. CTL, control. DAPI, 4′,6-diamidino-2-phenylindole. ( B ) Immunoblotting analysis of total, cytosolic (Cyto), and nuclear (Nuc) fractions of HEK293T cells with or without x-ray treatment (a dose of 4 Gy). ( C ) Immunoblotting analysis confirming the efficiency of ATR and ATM inhibitors (ATRi and ATMi). HEK293T cells treated with KU60019 (10 μM) or VE-821 (10 μM) for 24 hours and x-ray were subjected to immunoblotting analysis. ( D ) Immunoblotting analysis confirming the efficiency of DNA-PKcs inhibitor (DNA-PKcsi). HEK293T cells treated with NU7441 (2 μM) for 24 hours and x-ray were subjected to immunoblotting analysis. ( E ) Subcellular localization of FLAG-tagged YTHDF1 under the same conditions as (C) and (D). Lentiviral transduction was used for the expression of YTHDF1-FLAG. ( F ) Validation of ATR knockdown efficiency by immunoblotting in HEK293T cells. ( G ) Intracellular localization of YTHDF1 in control and ATR knockdown cells with x-ray treatment (a dose of 4 Gy). ( H ) Coimmunoprecipitation experiment confirming the interaction between ATR and YTHDF1. Lysates from HEK293T cells with or without x-ray treatment (a dose of 4 Gy) were immunoprecipitated with immunoglobulin G (IgG) or anti-ATR and then immunoblotted with anti-YTHDF1 antibody. IP, immunoprecipitation. ( I ) Intracellular localization of alanine mutants of YTHDF1 in HEK293T cells. A dose of 4-Gy x-ray irradiation was applied. ( J ) In vitro kinase assay using antibody-affinity–isolated ATR complex with purified YTHDF1 or YTHDF1-S182A. The phosphorylation was detected using a p-serine antibody (top). YTHDF1 and ATR were detected by Western blot (middle) and Coomassie blue staining (bottom). GFP, green fluorescent protein.
Article Snippet: Antibodies used in the experiments are as follows: YTHDF1 (1:2000; 17479-1-AP, Proteintech), γH2AX (1:2000; 16-202A, Sigma-Aldrich), ATM pS1981 (1:1000; ab36810, Abcam), ATM (1:5000; ab32420, Abcam), hemagglutinin (HA) (1:5000; AE008, ABclonal), ATR (1:1000; 13934, Cell Signaling Technology), ATR pS428 (1:1000; 2853, Cell Signaling Technology), DNA-PKcs (1:1000; ab70250, Abcam), DNA-PKcs pS2056 (1:1000; ab18192, Abcam), SF3B1 (1:3000; 27684-1-AP, Proteintech), SF3B3 (1:3000; 14577-1-AP, Proteintech), SRSF2 (1:1000; ab204916, Abcam), α-tubulin (1:5000; A6830, ABclonal), β-actin (ACTB) (1:4000; 23660-1-AP, Proteintech), lamin B1 (1:1000; 13435, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3000; 60004-1-Ig, Proteintech).
Techniques: Immunofluorescence, Control, Western Blot, Transduction, Expressing, Biomarker Discovery, Knockdown, Immunoprecipitation, Irradiation, In Vitro, Kinase Assay, Isolation, Purification, Phospho-proteomics, Staining
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