Fig. 2.

Analysis of Mef2A activity. (A) Basal mRNA expression of Mef2A, C and D in HEK293 cells, HUVECs and HMECs. Samples were analyzed by qPCR and normalized to β-actin (n = 3). (B) Mef2 luciferase reporter gene assay. HEK293 cells were transfected with the reporter plasmid 3xMef2-Luc and Mef2A together with either GFP as control or HDAC4, respectively. Luciferase activity was measured with luciferin and is illustrated relative to the GFP control (n = 8) (One sample t-test, *p < 0.05). (C) Co-immunoprecipitation of Mef2A and HDAC4. HEK293 cells were transfected with Mef2A-flag and flag-HDAC4-myc for 48 h. Cells were treated with or without H2O2 (100 μM, 30 min). IP was conducted with Protein G beads and IgG or myc tag antibodies. Western blots were stained for flag tag. (D) Mef2A luciferase reporter gene assay in HEK293 cells. Mef2A and HDAC4 were overexpressed together with Nox4 (WT-Nox4) or redox dead Nox4 mutant (RD-Nox4). Relative Mef2 activity is illustrated (n > 52) (Unpaired t-test, *p < 0.05). All bar graphs show means ± SEM.