*4.12. Western Blot Analysis*

Total cell lysates (50 μg proteins) from the different experimental conditions were subjected to 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes.

After regular blocking and washing, the membranes were incubated with specific primary antibodies over night at 4 ◦C.

For the determination of glucose receptors and tight junction levels: rabbit polyclonal anti-GLUT2 antibody (JJ20-21 Invitrogen, USA, diluted 1:500), rabbit polyclonal anti-SGLT1 antibody (Pa5-84085 Invitrogen, USA, diluted 1:200), and rabbit polyclonal anti ZO-1 antibody (GTX108587 GENETEX, Irvine, California, USA, diluted 1:200).

For the determination of glycolaldehyde-modified proteins (GA-modified proteins) levels: goat polyclonal anti-AGE antibody (Ab9890 Merck KGaA, Darmstadt, Germany, diluted 1:2000). Vinculin was used as the loading control (sc-25336 Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:200).

Donkey anti-goat (AP186P Sigma-Aldrich, Merck KGaA, Darmstadt, Germany, diluted 1:100,000), goat anti-mouse (sc-2005 Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:100,000), and goat anti-rabbit (12-348Sigma-Aldrich, Darmstadt, Germany, diluted 1:150,000) secondary antibodies HRP (Horseradish Peroxidase) were used in accordance with the manufacturer's instructions.

Protein bands were developed by the enhanced SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The chemiluminescent signal was acquired using ChemiDoc XRS+ System (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed by using Image J software (Version 1.50i, National Institute of Health, Bethesda, MD, USA)**.**
