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. 2014 Jun 16;111(26):E2646–E2655. doi: 10.1073/pnas.1323107111

Fig. 3.

Fig. 3.

RNF144A physically interacts with DNA-PKcs. (A) An IP-MS experiment identified DNA-PKcs as an RNF144A-interacting protein. HEK293T cells were transfected with an empty FLAG vector or FLAG-RNF144A. After 24 h, some cells were treated with NCS (300 ng/mL) for an additional 5 h. Cell lysates were harvested for FLAG-IP. Shown is a Sypro Ruby protein stain of the gel. The indicated bands were verified to be DNA-PKcs and RNF144A by MS. *Anti-FLAG antibody heavy chain and light chain. MS identified 66 peptides derived from DNA-PKcs (sequences presented in Table S1). (B and C) IP analysis shows that (B) only FLAG-RNF144A but not FLAG-RNF144B interacted significantly with endogenous DNA-PKcs in both untreated and MG132-treated (for 6 h) conditions and (C) this interaction could still be seen after DNase/RNase treatment. IB, immunoblot; WCL, whole cell lysates. HEK293T cells were transfected with an FLAG vector or FLAG-RNF144A. After 24 h, cell lysates were harvested for FLAG IP and Western blot analysis as indicated. A shorter exposed film (labeled Shorter exp.) of FLAG immunoblot after FLAG IP is also shown. (D) Western blot analysis confirmed an interaction between endogenous RNF144A and DNA-PKcs in U2OS and K562 cells. Cell lysates were harvested for endogenous RNF144A IP followed by Western blot analysis. (E) GST pull-down experiment shows that RNF144A directly bound to DNA-PKcs. Purified DNA-PKcs proteins (Promega) were incubated with purified GST or GST-RNF144A protein followed by Glutathione Sepharose pull-down and SDS/PAGE and Western blot analyses. (F and G) RNF144A–DNA-PKcs complex exists in both NCS and ADR treatment conditions. HEK293T cells were transfected with FLAG or FLAG-RNF144A. After 24 h, cells were treated with NCS (300 ng/mL) or ADR (5 μM) for an additional 5 h. Cell lysates were then harvested for FLAG IP and immunoblotting. (H) Different truncated RNF144A fragments were transfected to HEK293T, and cells were treated with MG132 for 6 h; the FLAG IP was performed as above. This deletion analysis indicates that RNF144A (amino acids 173–252) interacted well with DNA-PKcs. (I) A scheme of human RNF144A and its domains. Ctrl, control.