Whether or not there are molecular differences, at the intra- and
Whether or not there are molecular differences, at the intra- and extracellular level, between aortic dilatation in patients with bicuspid (BAV) and those with a tricuspid aortic valve (TAV) has remained controversial for years. HSP27 and dually phosphorylated HSP27 (S78/S82) were investigated in an extended patient cohort (n=15) using ELISA. Total HSP27 was significantly lower in BAV compared to TAV patients (p=0.03), with no correlation in levels of phospho-HSP27 (S78/S82) (p=0.4). Western blots analysis showed a trend towards lower levels of phospho-HSP27 (S78) in BAV patients (p=0.07). Immunohistochemical analysis revealed HSPB1 that differences in HSP27 occur in the cytoplasma of VSMCs and not extracellularly. Alterations in HSP27 may give early evidence for intracellular differences in aortic aneurysm of patients with BAV and TAV. Whether HSP27 and the defined phosphoproteins have a specific role in BAV associated aortic dilatation remains to be elucidated. for 5 478-43-3 supplier min at 4C, and the supernatant was saved as protein extract. Protein concentrations were obtained using BCA assay done in triplicate. 2-D Gel Electrophoresis IEF was carried out using a Protean IEF cell (BioRad, Hercules, CA) according to the manufacturers protocol. For the first dimension of separation 100ug of total protein was applied. Immobilized pH gradient (IPG) Dry Strip (gradient pH 3C10, 18 cm, GE Healthcare, Piscataway, CA) were actively rehydrated at 50 V for 10 h to enhance protein uptake, then subjected to the following conditions using a rapid voltage-ramping method: 100 V for 25 Vh, 500 V for 125 Vh, 1000 V for 250 Vh, and 8000 V for 85 kVh. For SDS-PAGE, IPG strips were incubated for 10 min in equilibration buffer (50 mmol/L Tris-HCl [pH 8.8], 6 mol/L urea, 30% vol/vol glycerol, 2% wt/vol SDS) supplemented with 10 mg/mL DTT, followed by a 10-min incubation in equilibration buffer supplemented with 25 mg/mL iodoacetamide, then rinsed once with SDS-PAGE buffer (25 mmol/L Tris, 192 mmol/L glycine [pH 8.3], 0.1% wt/vol SDS). IEF strips were then embedded in a 4% acrylamide stacking gel, and the proteins were resolved by SDS-PAGE using a Protean II XL system (BioRad, Hercules, CA). Electrophoresis was carried out at 50 V for 30 min, followed by 150 V for 7.5 h. In addition, a part of the 2DE was done in the Andersons ISODALT system for the simultaneous analysis of 20 samples (instruments produced in the workshop of the former Basel Institute for Immunology, Switzerland) . The first dimension of separation was a pH gradient nonlinear 3-10 and the second dimension 11-19% SDS-PAGE (Figure 1). Silver staining of the 2-D gel was performed according to the protocol of Shevchenko, which is compatible 478-43-3 supplier with subsequent protein analysis by mass spectrometry . Image analysis was carried out using PDQuest (BioRad, Hercules, CA). Figure 1 Representative 2-D gel (pH gradient 3-10, 11-19% SDS-PAGE) of an aortic aneurysm sample from a patient with a TAV. Magnified 2-D pattern of five BAV (A1-A5) and five TAV (B1-B5) are shown. The arrows mark the spot that is significantly lower expressed … Assessment of phosphorylation status: Dephosphorylation and Phosphostaining The aortic sample of a patient with a TAV was dephosphorylated with alkaline phosphatase using the 478-43-3 supplier following protocol: an aliquot of the aortic protein extract was divided into two equal volumes (100 g each). Alkaline phosphatase reaction buffer (10x NE Buffer 3, New England Biolabs, Ipswich, MA) was added to each sample, 2 L (10000 U/ml) of calf intestinal alkaline phosphatase (New England Biolabs, Ipswich, MA) was added to one sample, and then deionized distilled 478-43-3 supplier water was added to both samples to total volumes of 25 l. Samples were mixed well and incubated at 37C for 18 h. The reaction was stopped with isoelectric focusing (IEF) buffer, and samples were frozen at -80C until further analysis. The dephosphorylated and untreated samples were separated by 2DE as above except the 2DE gel underwent a phosphostaining procedure using Pro-Q Diamond phosphoprotein gel stain (Molecular Probes, Invitrogen, Carlsbad, CA) according to the manufacturers protocol. Fluorescent staining of the SDS-polyacrylamide gels was performed by fixing the gels in 50% methanolC10% acetic acid overnight, washing with three changes of deionized distilled water.