FIGURE 3.

Mapping of ACTN4- and GR-interacting domains. A, top panel, schematic of ACTN4 functional domains (CH, calponin homology domain; CaM, calmodulin), the nuclear receptor-interacting motif, LXXLL (amino acids 84–88), and the C-terminal EF-hand calcium binding domains. The FSGS-linked K255E, T259I, and S262P mutants are indicated. Bottom panel, HeLa cells were transfected with an HA-GR expression plasmid. Whole cell lysates were incubated with immobilized GST and GST-ACTN4 and its variants. GST pulldown assays were performed as described for Fig. 2B. Pulldown fractions were analyzed by Western blotting with anti-HA antibodies. The expressions of GST fusion proteins were visualized by Coomassie Blue staining. B, top panel, schematic of GRα functional domains (NTD, N-terminal domain; DBD, DNA-binding domain; H, hinge domain; LBD, ligand-binding domain). Bottom panel, HeLa cells were transfected with HA-GR truncated mutants. Whole cell lysates were incubated with immobilized GST and GST-ACTN4. GST pulldown assays were performed as described. Dex, dexamethasone.