The Complexity of Sporadic Alzheimer’s Disease Pathogenesis: The Role of RAGE as Therapeutic Target to Promote Neuroprotection by Inhibiting Neurovascular Dysfunction
Figure 3
Aβ enhances TXNIP translocation to the plasma membrane. (a) RBE4 cells were maintained 5 days in differentiation medium (F10/MEM, 2.5% FCS, hydrocortisone 14 μM, Hepes 10 mM, bFGF 1 μg/mL). RBE4 cells were stimulated for 45 min with Aβ1-42 (3 μM). Subcellular fractions were obtained using a cell fractionation kit (Biorad) according to the manufacturer instruction. The presence of TXNIP, RAGE, VE-cadherin, and histone H3 were analyzed by western blotting. (b) RBE4 cells were maintained as described in (a) and stimulated for 45 min with Aβ1-42 (3 μM). Cells were fixed in PBS containing 4% PFA and permeabilized 10 min in PBS 0.1% Triton X-100. Cells were maintained 1 h in blocking solution (PBS 3% BSA) at room temperature and then incubated over/night at 4°C with a rabbit anti-TXNIP polyclonal antibody (Invitrogen) and a mouse anti-VEcadherin monoclonal antibody (Santa Cruz biotechnology) in blocking solution. Cells were washed 3 times for 15 min with PBS and incubated with the appropriate secondary antibody. Nuclei were stained with Hoecst. Immunofluorescence was analyzed by a confocal microscopy (Zeiss). These data are representative of 3 independent experiments.
