Figure 2: ARD1 regulates the stress response of Hsp70.
From: ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation
(a) ARD1 binds to Hsp70 but not to Hsc70. FLAG-ARD1 was precipitated from HEK293T cells, and co-precipitation of Myc-Hsp70 or Myc-Hsc70 was assessed by western blotting. (b) ARD1 is required for stress-induced Hsp70 acetylation. SH-SY5Y cells were transfected with ARD1 siRNA. After treatment of 1 mM H2O2 for 1 h in SH-SY5Y cells, GFP-Hsp70 was precipitated, and its acetylation level was assessed with an anti-Lys-Ac antibody. (c) ARD1 regulates the acetylation and co-chaperone bindings of Hsp70. HEK293T cells were transfected with ARD1 siRNA. After cells were treated with 1 mM H2O2 for 1 h, endogenous Hsp70 was precipitated using an Hsp70 antibody, and then its acetylation level and co-chaperone binding were analysed by western blotting. (d) ARD1 acetylates Hsp70 but not Hsc70 in vitro. GST-ARD1, GST-Hsp70 and GST-Hsc70 recombinants were subjected to an in vitro acetylation assay for 1 h, and acetylation levels of recombinants were assessed by western blotting using an anti-Lys-Ac antibody.
