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. 2021 Jul 9;49(14):8145–8160. doi: 10.1093/nar/gkab585

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Tpk1 effect on NHEJ is associated with Dun1 and Nej1. (A, B) Chromosomal DSB repair efficiency in tpk1 and dun1 (or nej1) double mutants (A) and their single mutants, as well as in the overexpression (B) of NEJ1 or DUN1 in tpk1 mutants. (C) Nej1 protein expression at different time points post BLM-induction in HA-tagged Nej1 in wild-type (WT) and tpk1 mutants along with yeast (Sc) GAPDH control (Ctrl). (D) Immunoblot (IB) showing the native expression of chromosomally FLAG-tagged Tpk1 and/or Nej1 in whole cell lysates (WCL) and their physical association with proteins (filtered at q ≤ 0.01 false-discovery rate, FDR) involved in DNA repair-related processes (Supplementary Table S3); PPIs, protein-protein interactions. (E, F) TAP-tagged Tpk1 immunoprecipitated (IP) with calmodulin beads from the cell extracts of HA-tagged Nej1 (wild-type, mutagenized; E) or Dun1 (F), treated with or without bleomycin (BLM) in the presence or absence of Tpk1, was IB’ed with anti-TAP or anti-HA antibody. The expression of TAP or HA-tagged proteins in whole cell or IPed lysates were IB’ed with anti-TAP or anti-HA antibody as controls (ctrl). Molecular masses (kDa) of marker proteins are shown in C, E and F. Data in panels A and B represent mean ± SD (n = 5 biological replicates; *P ≤ 0.05 by Student's t test).