Fig. 5: MED23 G382F mutation impaired transcriptional activity of Elk-1.

a Close-up view of MED23-Elk-1 (374–384) interface with MED23 G382 residue colored in deep pink. G382 was mutated in Phenylalanine using the ChimeraX rotamers tool, potentially obstructing the space occupied by F378-Elk-1. b Elk-1 TAD binding is disrupted by MED23 G382F mutation. GST and GST Elk-1^8P (308–428) were immobilized on GST-Trap agarose and incubated with recombinant MED23 or MED23 G382F. Bound proteins were eluted with Laemmli sample buffer, resolved by SDS-PAGE, and visualized by Coomassie Blue staining. The molecular weight marker (in kDa) is indicated on the left. 1: MED23 WT and G382F input 1/40; 2: GST control and 3: phosphorylated GST Elk-1^8P (308–428). c Defective Elk-1 TAD activity in MED23^-/- MEFs. MED23^-/- MEFs were transfected with a 5 × Gal4-E1B-TATA-luciferase reporter construct and a plasmid encoding either Gal4-Elk-1^8P (308–428) or Gal4-E1A activation domain (13S CR3) with empty (0, circle), MED23 WT (square) and MED23 G382F (triangle) expression plasmids as indicated. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are representative of three independent experiments and are presented as mean values ± SD. d MED23^-/- MEFs stably expressing MED23 WT (MEF WT, circle) or MED23 G382F (MEF G382F, square) were generated and transfected as in Fig. 5c. DNA binding domain of Gal4 (G4) was used as control. Data are presented as mean values of two biological replicates ± SD. e Interaction between Elk-1 and MED23 is necessary in serum induction of Egr1. Time course of EGR1 protein expression after serum induction in MED23^-/- MEFs, MEFs^+/+, and MED23^-/- MEFs stably expressing MED23 WT or MED23 G382F. Cell lysates at the indicated minutes after serum addition to serum-starved cells were analyzed by Western blot with anti-MED23, anti-EGR1, and anti-Actin antibodies as indicated. The star indicates an unknown band. A molecular weight marker (in kDa) is indicated. f qRT-PCR analysis of immediate early genes transcription in MED23^-/- MEFs stably expressing MED23 WT or MED23 G382F. The expression was normalized to GAPDH mRNA expression. Data represent fold change of Egr1, Egr2, Egr3, and Fos in MED23 WT relative to MED23 G382F MEFs. Data are presented as mean values (n = 4) of three biological replicates ± SD. Source Data are provided as a Source Data file.