Protein Expression Analysis by Western Blot

Western blot analysis was performed as previously described [19 (link)]. Primary antibodies against occludin (c-term), ZO-1, and claudin-5 and secondary antibodies, Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit, were purchased from Thermo Fisher Scientific. To detect the formation of AGE products with MG-modification, mouse anti-methylglyoxal monoclonal antibodies (STA-011; Cell Biolabs, San Diego, CA, USA) were used. Primary antibodies against p-mTOR, t-Akt, p-Akt, t-S6K1, p-S6K1, HIF-1α, COXIV, VDAC, and Parkin (Prk8) were purchased from Cell Signaling Technology (4695S, Danvers, MA, USA). Primary antibodies against t-mTOR and occludin (E-5) and the secondary antibody donkey anti-rabbit IgG-FITC were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody against LC3B was purchased from Sigma Aldrich. Primary antibodies against Glo-1 and Glo-2 were purchased from Abcam. To counterstain the total protein in the gel, Coomassie Brilliant Blue staining was used as previously described [8 (link)].