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. 2018 Aug 14;33(1):1138–1150. doi: 10.1096/fj.201800336R

Figure 5.

Figure 5

Truncated Raf1 preferentially binds DNA-PK and alters DNA-PK phosphorylation: A) PLA for endogenous Raf1 and DNA-PK in HEK cells. Interactions between Raf1 and DNA-PK are shown in red with nuclei (blue) counterstained with DAPI. B) PLA for FLAG and DNA-PK in HEK cells expressing FLAG-tagged Raf1-fl or Raf1-tr. Interactions between FLAG-tagged Raf1-fl or Raf1-tr and DNA-PK are shown in red with nuclei (blue) counterstained with DAPI. Images are representative of experiments run in triplicate. C) Quantitation of Raf1-fl and Raf1-tr interactions with DNA-PK. A minimum of 150 cells per group were analyzed. D) Western blot of DNA-PK Ser 2056 and Thr 2609 phosphorylation, and total DNA-PK in HEK cells expressing empty vector, Raf1-fl, or Raf1-tr exposed to vehicle (−) or 0.05 mg/ml bleomycin (+) for 2 h. β-actin was used as a loading control. Blot is representative of experiments run in quadruplicate. E) Quantitation of phosphorylated DNA-PK normalized to empty vector exposed to bleomycin (Bleo). The results are shown as mean ± sem. Scale bars, 50 µm.