Fig. 7

Fig. 7

A Representative 2D silver stain of human LV tissue. B Selected spot areas 1 to 6 were analyzed by liquid chromatography followed by mass spectrometry (LC-MS). Peptide counts uniquely assigned to ventricular (v) ELC or vRLC are shown for 6 independent samples. The mean percentage of the protein coverage of vELC and vRLC are given above (table inset). Data are mean ± SD. C Schematic illustration of identified ELC or RLC-phosphorylation sites and deamidated amino acid residues detected in human LV tissue (n = 6; DCM: n = 4; Ctrl: n = 2). D Validation of polyclonal rabbit antibody against human ELC (Biozol, GeneTex, ZF127578) by immunoblot (IB) of different amounts of human LV protein. No cross reactivity with vRLC was detected. E Quantification of relative ELC protein expression illustrated as averaged intensity normalized to total ELC protein. ELC expression intensities were plotted against the amount of human heart protein. Exposure time was varied from 0.25 to 5 min. For 2 min exposure R = 0.9383 and P < 0.0001 was calculated by two-tailed Spearman Rank Correlation (n = 5). F Validation of in-vitro dephosphorylation of human LV tissue. After protein isolation samples were split and half of each sample was treated with phosphatase inhibitor (Native), while the other half was incubated with phosphatase (Dephospho). Proteins of the same aliquot were analyzed either by ELC or RLC 2D immunoblot (IB) (left panel) or IB against phospho-cardiac troponin I (TnI) (right panel). The expression of total cardiac TnI was used as loading control. The same aliquots were used for Pro-Q® Diamond Phosphoprotein Stain to detect phosphorylated protein species in 2D IB (Supplementary Fig. 9).