Fig. 1: Generation and validation of HaloTagged core NHEJ proteins in U20S cells.
From: Single-molecule imaging reveals the kinetics of non-homologous end-joining in living cells
A Graphical depiction of the long-range XLF-mediated NHEJ synaptic complex and HaloTag (based on PDB: 7NFC and 6U32)13,44. B Western blots of U2OS cells expressing HaloTagged NHEJ factors and parental U2OS cells probed with antibodies against Ku70 (top), XRCC4 (middle), and XLF (bottom). C Representative images of 20 living cells expressing HaloTagged NHEJ factors and Halo-Rif1 labeled with JFX650 HaloTag ligand in the presence or absence of zeocin (Scale bar = 10 µm, also see Supplementary Fig. 1B, C). D Clonogenic survival assay of U2OS cells expressing HaloTagged NHEJ factors, parental U2OS cells, and U2OS cells with DNA-PKcs knocked out after challenge with Zeocin (N = 4, 3 technical replicates per biological replicate, Mean ± S.D.). E Fluorescence imaging of 2 clones of each HaloTagged NHEJ factor labeled with JF650 HaloTag ligand and separated on an SDS-PAGE gel. F Quantification of the total protein abundance based on recombinant 3X FLAG-HaloTag labeled with JF646 and cell lysates from a specific number of U2OS using in-gel fluorescence intensity values after applying a correction factor from western blots (see Supplementary Fig. 1E, F, N = 3 biological replicates, Mean ± S.D.). Source data are provided in the Source Data file.
