Fig. 4. Increased ATF2 expression in FDC hearts.

a Total RNA was isolated and reverse transcribed from control hearts (n = 3, biological replicates), from the hearts of the patients with the FBXO32 mutation (Family A, n = 4, technical replicates), and from idiopathic dilated hearts (n = 3, biological replicates). mRNA expression for enzymes playing roles in transcription, protein stability, and chromatin regulation was measured by qPCR using the RT² Profiler PCR Array (Qiagen). Transcripts significantly differentially regulated between the FBXO32 mutant hearts and IDC hearts are shown as fold differences over controls, corrected for 5 housekeeping genes. Significant p values calculated using a Student’s t test are shown. b Western blot analysis performed from human heart lysates prepared from control hearts (n = 3), the two hearts with the FBXO32 mutation (FDC), and idiopathic dilated hearts (IDC) (n = 4) using a specific anti-ATF2 antibody. GAPDH expression was measured in parallel for loading control. c Quantitation of (b) using Adobe photoshop CS6-program. Results are expressed as average ± SD and are normalized against GAPDH. d Representative immunoblots showing ATF2 protein expression in untransfected HEK293 cells or in cells overexpressing mutant-FBXO32 or WT-FBXO32. e Quantitative analysis of (d). Results are normalized against GAPDH and expressed as average ± s.e.m., n = 5 independent experiments. The statistical significance was determined by Student’s t-test. Exact p values are shown. Ns non-significant.