Fig 1: RAD54 phosphorylation occurs at T700 (pRAD54-T700) in cells in a TLK1 dependent manner and increases post IR recovery. (A) pRAD54-T700 antibody specificity characterized to detect T700 site. (B) pRAD54-T700 decreases by 60% after TLK1 inhibition. A parallel blot was probed for pNek1-T141 and Nek1. (C) Quantification of relative level of pRAD54-T700 phosphorylation level from panel B. (D) pRAD54-T700 is initially lost at 0.5hr, but increases post IR recovery (4 and 12 h). (E) Phosphorylated RAD54-T700 and RAD51 association increases in nucleus post IR induction in S-phase synchronized cells. RAD51 and p-RAD54-T700 probed in nuclear fraction of S-phase cells post DNA damage recovery as indicated. ORC2 is shown as chromatin associated nuclear loading control. Lane 1–2, 3–4, 5–6 indicates nuclear (Nuc) fractions from S-synchronized cells without or with IR (10 Gy) and allowed recovery period of 0, 4, 12 h, respectively. Lanes 7 and 8 indicates asynchronous (Asyn) cells. (F) Quantification of pRAD54-T700 in S-phase nuclear fraction is shown. (G) Quantification of RAD51 in S-synchronized nuclear fraction is shown. *P< 0.05; **P< 0.01; ***P< 0.001; ****P< 0.0001; ordinary one-way ANOVA followed by Tukey's post-hoc analysis was performed for multiple group comparison.