Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex
J. Clin. Invest. Hui-Hua Li, et al. 114:1058 doi:10.1172/JCI22220 [Go to this article.]

Figure 2
Mapping the interaction domains of atrogin-1, α-actinin-2, and calcineurin A. (A) The residues of atrogin-1 required for binding to α-actinin-2 and calcineurin A were determined with GST pull-down assays. GST–atrogin-1 fusion proteins were purified and analyzed for expression (top). The ability of the truncated atrogin-1 fusion proteins to bind to calcineurin A (expressed in COS-7 cells as a GFP fusion) and α-actinin-2 (expressed as an HA fusion) was analyzed by blotting with antibodies against HA (middle) and GFP (bottom). (B) GST–calcineurin A was affinity-purified and analyzed by blotting with antibody against GST (top). The pull-down (middle) and input (bottom) fractions of COS-7 cell extracts expressing the indicated Myc-tagged atrogin-1 truncations were immunoblotted with antibodies against Myc. (C) Schematic representation of atrogin-1 truncations that interact with α-actinin-2 and calcineurin A. F-box, F-box domain; NLS, nuclear location sequence; PDZ, PDZ domain. (D) The region of calcineurin A involved in binding to atrogin-1 was analyzed in pull-down assays. GST–calcineurin A fusion proteins were affinity purified and analyzed by blotting with GST antibody (top). The ability of the various calcineurin A fusion proteins to bind to atrogin-1 expressed as a Myc-tagged fusion in COS-7 cells was analyzed by blotting with Myc antibody (bottom). (E) Schematic representations of calcineurin A residues that bind to atrogin-1. CnB, calcineurin B–binding domain; CaM, calmodulin-binding domain; AID, autoinhibitory domain.