Stage 3/2009
Goal:
1. Isolation and analysis of lipid raft/caveolae enriched membrane fractions and cytosolic fractions from normal/pathological tissue.
2. Identification of mechanisms and signaling pathways in which MHGB1 is involved in normal and pathological conditions.
Activities:
1.1 Tissue solubilization in non-ionic detergents and isolation of lipid raft/caveolae by ultracentrifugation in sucrose gradient.
1.2 Separation of peptides in bidimensional polyacrylamid gels (2D SDS-PAGE) on pH stripes immobilized in gel of 24 cm, with values between pH 4-7 and 6-9 and isoelectrical focusing. Data analysis with specialized software.
2.1 Analysis of signaling pathways (Hsp27, Akt, Hf-kB) in order to establish the mechanisms of HMGB1 activity in normal and pathological conditions.
Results:
Figure 1 - Ultra-centrifugation tubes showing fraction 5 in normal (N), folic acid deficient (D) and hyperlipidemic/folic acid deficient (H) diet.
Figure 2 - Biochemical protein concentration determination in the 12 fractions obtained from the ultra-centrifugation for each type of hamster samples (N,D,H). Fraction 5 has been corelated with the lipid rafts.
Figure 3 - Biochemical cholesterol concentration determination in the 12 fractions obtained from the ultra-centrifugation for each type of hamster samples (N,D,H). Fraction 5 has been corelated with the lipid rafts.
Figure 4 - Caveolin 1 expression in the 12 fractions obtained after the ultra-centrifugation visualized by immunotransfer.
Figure 5 - Electrophoretic profile of the proteins from the fractions 4,5 and 6 combined, visualized by silver nitrate
Figure 6 - Bidimensional polyacrylamide gel (2D SDS-PAGE) from lipid rafts with the combined pH domain between 4 and 9.
Figure 6 - Protein spots under/overexpressed in folate decicient diet hamster.
Figure 6 - Protein spots under/overexpressed in hyperlipidemic diet hamster treated with fluvastatin.
Figure 6 - Expression of HSP proteins in endothelial cells loaded with hiperlipidemic ser.
Figure 6 - Expression of NF-kB in endothelial cells in normal and pathological conditions.
Figure 6 - Expression of Akt and RAGE in U937 macrophage cell line; a: immunoblotting of Akt and RAGE, b: values obtained from densitometry of Akt and pAkt normalized with beta-actin.
