Gtx113381

To extract total protein from lenses, lenses were homogenized in lysis buffer composed of protease and phosphatase inhibitor cocktails for 1 h at 4 °C. The extracted protein concentration was quantified by BCA protein assay (Pierce), subjected to 7.5–10% SDS-Tris glycine gel electrophoresis, transferred to polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). The membrane was incubated in 5% non-fat skim milk in with Tween-20 buffer (10 mmol/L Tris, 150 mmol/L NaCl, and 0.1% Tween-20, pH 7.4) for 1 h. Primary antibodies: AKR1B1, MMP9, vimentin (GTX113381, GTX100458 and GTX100619, respectively, Genetex, Irvine, CA, USA), N- and E-cadherin (610920 and 610182, respectively, BD Biosciences, Franklin Lakes, NJ, USA), Phosph-AMPK α1,2^T172 (44-1150G, Thermo Fisher Scientific, Eugene, OR, USA), and SOD2 (acetyl K68) (ab137037, Abcam, Eugene, OR, USA) were diluted in PBST at 1:2000 dilutions and incubated at 4 °C overnight. Peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:10,000) were used. The result was visualized using ECL-Plus detection kit (GE Healthcare) and exposed to film. The developed films were scanned (photo scanner 4490, Epson, Long Beach, CA, USA), analyzed using NIH image densitometry analysis software (National Institutes of Health, Bethesda, MD, USA).

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.