Sc 8230

By using ice-cold RIPA buffer the total protein was isolated from hippocampal tissues. BCA Protein Assay Kit (Thermo Fisher Scientific, USA) was used to measure the protein concentrations. A nitrocellulose membrane (Millipore Co., USA) was used for immuno-blotting where the protein samples (30 μg) were separated using SDS–polyacrylamide gel electrophoresis and then transferred to. For 1 h the membrane was blocked with 5% skim milk in Trisbuffered saline (150 mM NaCl, 0.1% Tween 20, 20 mM Tris, pH 7.4). Then the proteins were detected by incubation with primary antibodies against HMGB1, RAGE at 4 °C overnight. Later the membrane was washed for 3 times in Tween 20 + PBS and was incubated by the horseradish peroxidase-conjugated secondary antibodies for 2 h. The immuno-blots were visualized by a Millipore ECL Western Blotting Detection System and the variables protein expression levels were normalized to those of β-actin.
The following antibodies were used: anti-β actin (1:2,000, sc-130657, Santa Cruz Biotechnology), anti-HMGB1 (1:1,000, ab18256; Abcam), anti-RAGE antibody (1:2,000, sc8230; Santa Cruz), anti-NLRP3 antibody (1:2,000, sc-66846, Santa Cruz Biotechnology), anti-ASC antibody (1:2,000, sc-22514-R, Santa Cruz Biotechnology), pro and cleaved Caspase1 antibody (1:2,000, sc-514, Santa Cruz Biotechnology) and pro and mature IL-1β (1:1,000, AF-401-NA, R&D Systems).

Primary antibodies for RAGE and nitrotyrosine (sc-8230 and sc-32757, respectively, Santa Cruz, CA, USA), E- and N-cadherin (610182 and 610920, respectively, BD Biosciences, San Jose, CA, USA), AKR1B1, MMP9, vimentin (GTX113381, GTX100458, and GTX100619, respectively, Genetex, Irvine, CA, USA), SOD2 (acetyl K68) (ab137037, Abcam, Eugene, OR, USA), and Phosph-AMPK α1,2^T172 (44-1150G, Thermo Fisher Scientific, Eugene, OR, USA) were used for immunofluorescence staining.

Experimental drugs including fructose and dimethyl sulfoxide (DMSO) were all obtained from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA). D3T (Abcam, ab141925) was obtained from Abcam (Abcam Chemical Co., Cambridge, MA, USA). Primary antibodies, nitrotyrosine, and RAGE (sc-32757 and sc-8230, respectively, Santa Cruz, San Jose, CA, USA), N-cadherin and E-cadherin (610920 and 610182, respectively, BD Biosciences, San Jose, CA, USA), AKR1B1, MMP9, and Vimentin (GTX113381, GTX100458 and GTX100619, respectively, Genetex, Irvine, CA, USA), Phosph-AMPK α1,2^T172 (44-1150G, Thermo Fisher Scientific, Eugene, OR, USA), and SOD2 (acetyl K68) (ab137037, Abcam, Eugene, OR, USA), and secondary antibodies, Alexa Fluor 568 and 488 goat anti-rabbit or mouse, and Alexa Fluor 488 donkey anti-goat, were used to stain the LECs. In addition, the VECTASHIELD^® HardSet™ antifade mounting medium with DAPI (H-1500, Vector, Burlingame, CA, USA) was used to mount the stained epithelial cell sections and counterstain them.