Figure 5: Unbound XRCC1 is an HSP90 client protein.
From: HSP90 regulates DNA repair via the interaction between XRCC1 and DNA polymerase β

(a) HSP90 interacts with XRCC1 but not other BER-related proteins. WCL were prepared from proliferating T98G/MPG/Polβ-KD cells expressing EGFP, Flag-Polβ(WT) or Flag-Polβ(TM) (left panel). XRCC1 and other BER-related proteins were immunoprecipitated with an HSP90 antibody and examined by immunoblot as shown (right panel). (b) HA-HSP90 interacts with XRCC1 in Polβ-KO MEFs, not in WT MEFs. WT (92TAg) and Polβ-KO (88TAg) MEFs, after expression of HA-HSP90, were probed for HSP90-interacting proteins by IP of the expressed HA-HSP90 via the N-terminal HA epitope tag and probing for XRCC1 by immunoblot. Input HA-HSP90 is also shown. (c) HSP90 inhibitor 17-AAG treatment induces the degradation of XRCC1 in cells lacking Polβ (LN428/Polβ-KD cells and MEFs), as indicated: immunoblot of WCLs from stable LN428 cell lines expressing Flag-Polβ(WT) or Flag-Polβ(TM) and from stable LN428/XRCC1-KD cells expressing Flag-Polβ(WT) (shown in top panel) and immunoblot of WCLs from WT (92TAg) and Polβ-KO (88TAg) MEFs (shown in bottom panel) after exposure to 17-AAG at the concentrations indicated. The immunoblot image depicts the levels of XRCC1, Flag-Polβ and PCNA, before and after treatment with 17-AAG. A representative immunoblot image is shown. For quantified results, see Supplementary Fig. 5D,E. (d) Cycloheximide-enhanced degradation of XRCC1 in LN428/Flag-Polβ(TM) cells treated with 17-AAG is protected by MG132: (top panel) immunoblot of WCLs from stable LN428 cell lines expressing Flag-Polβ(TM) after exposure to 17-AAG (0 or 10 μM, as indicated) and cycloheximide (Cyclo) for the times indicated. A representative immunoblot image is shown. (Bottom panel) Immunoblot of WCLs from stable T98G/Polβ-KD cell lines expressing EGFP after exposure to 17-AAG (10 μM) and Cyclo or Cyclo+MG132 for the times indicated. A representative image is shown. For quantified results, see Supplementary Fig. 5F. (e) HSP90 knockdown induces the degradation of XRCC1 in LN428/Flag-Polβ(TM) and LN428/MPG/Polβ-KD/Flag-Polβ(TM) cells: immunoblot of WCLs from LN428 cell lines expressing Flag-Polβ(TM) or LN428/MPG/Polβ-KD cells expressing Flag-Polβ(TM) after lentiviral-mediated expression of GFP or HSP90-specific shRNA, as indicated. A representative immunoblot image is shown.