Fig. 4: Bufalin selectively binds to Arg394 (R394) of ERα.

a Bufalin was docked into the binding pocket of ESR1 using the Glide SP protocol. b The docked structure of the ESR1-Bufalin complex was then subjected to 100 ns of molecular dynamics simulations using Amber, followed by MM-GBSA energy calculations and residue energy decomposition. The top 10 residues from the MM-GBSA residue energy decomposition scoring. c The top 10 residues selected for alanine scanning mutagenesis. Error values were derived from energy snapshots based on 100 frames uniformly extracted from the last 100 ns of the MD simulation trajectory. d 293 T cell was transfected with ERα wild type (WT) plasmid or mutant plasmid, after transfected 48 h, the cell lysates were incubated with Biotin-Bufalin at 4 °C overnight, followed by pulling-down with streptavidin magnetic beads. The proteins bound to the magnetic beads were separated by SDS-PAGE, followed by western blot using ERα antibody. e 293 T cells were transfected with ERα WT or mutant plasmid, followed by treatment with Bufalin for 48 h. The ERα protein levels were measured by western blot. f 293 T cells were transfected with Flag-ERα WT or R394A mutant plasmid and HA-Ub plasmid, and then subjected to Bufalin for 48 h, followed by treatment with MG-132 (10 μM) for 10 hours before harvest. Then the cells lysates were subjected immunoprecipitation with anti-Flag antibodies and blotted with anti-HA antibodies.