G box mini multi fluorescence and chemiluminescence imaging system

The dissected liver tissues were subjected to nuclear protein extraction and EMSA using an extraction kit (NE-PER Nuclear and Cytoplasmic Extraction Kit, ThermoFisher Scientific, Waltham, MA, USA) and a commercial EMSA assay kit (LightShift^TM Chemiluminescent EMSA Kit, ThermoFisher Scientific, Waltham, MA, USA), respectively, according to the manufacturer’s instructions [43 (link),44 (link),45 (link)]. Oligonucleotides for the detection were as follows: 5′-AGTTGAGGGGACTTTCCCAGGC for NF-κB. The protein/DNA complexes were visualized and quantified using a G:BOX mini multi-fluorescence and chemiluminescence imaging system (Syngene, Frederick, MD, USA) after incubation with enhanced chemiluminescence Western blotting reagents.

Whole steps of tissue protein extraction, cell lysate preparation, SDS-PAGE, Western blotting and quantitative calculation were performed as reported procedures [32 (link),33 (link)]. Interesting molecules corresponding to the indicated antibodies were cyclin D1 (sc-56302), β-catenin (sc-133240), HMGB1 (sc-135809), RAGE (sc-74535), Akt (sc-5298), phospho-Akt (sc-293125), SLC7A5 (sc-374232), Smad2/3 (sc-398844), phospho-Smad2/3 (sc-11769) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), GLUT1 (ab115730), PKM2 (ab137852), SLC1A5 (ab84903), glutaminase (ab150474), CD62P (ab59738), MPO (ab65871) (Abcam, Cambridge, MA, USA) and Glyceraldehyde-3-Phosphate Dehydrogenase (AF5718, GAPDH) (R&D Systems, Minneapolis, MN, USA).The proteins of interest were visualized and quantified using a G:BOX mini multi-fluorescence and chemiluminescence imaging system (Syngene, Frederick, MD, USA).