Anti tonebp

Total proteins from retinas of wild-type C57BL/6 male mice and sex/age matched D2.B6-Ins2 Akita diabetic mice, or from HMVECs and HAECs were isolated, electroblotted, and incubated with the following primary and secondary antibodies: (1) goat polyclonal anti-COX-2 (cat. N° sc-1747, dilution 1:500, Santa Cruz Biotechnologies, Santa Cruz, CA USA), secondary antibody mouse anti-goat IgG-HRP (cat. N° sc-2354, dilution 1:5000, Santa Cruz); (2) mouse monoclonal anti-AQP1 (cat. N° sc-55466, dilution 1:600, Santa Cruz), secondary antibody goat anti-mouse IgG-HRP (cat. N° sc-2005, dilution 1:5000, Santa Cruz); (3) mouse monoclonal anti-β-actin (cat. N° A5441, dilution 1:5000, Sigma, St. Louis, MO, USA), secondary antibody goat anti-mouse IgG-HRP (cat. N° sc-2005, dilution 1:5000, Santa Cruz); (4) rabbit polyclonal anti-TonEBP (cat. N° sc-13035, dilution 1:500, Santa Cruz), secondary antibody goat anti-rabbit IgG-HRP (cat. N° sc-12304, dilution 1:10000, Santa Cruz), as previously described [25 (link)].

Cytoplasmic as well as nucleus-specific proteins in the PVN and SON were isolated by using the Nucleus and Cytoplasmic extraction kit (Thermo Scientific, Catalogue No. 78833), according to the manufacturer’s instruction. Briefly, protein concentration was measured by the Bradford dye-binding assay (Bio-Rad, Hercules, CA, USA, Catalog No. 5000006), and then, equal amount of proteins from each sample were separated by SDS-PAGE, transferred onto the PVDF membranes by electrophoretic transfer. The membrane was blocked with 5% non-fat skim milk in TBS-tween, and then incubated with anti-TonEBP (Santa Cruz, Catalog No. sc-398171; 1:1000 dilution). The membrane was incubated with HRP-conjugated mouse secondary antibody (Cell signaling, Danver, MA, USA, Catalog No. 7076; 1:3000 dilution), and the immunoreactive signals were detected by Chemiluminescent detection reagent (Thermo Scientific, Catalog No. 34095). Protein density was normalized using an anti-Hsc70 antibody (Rockland, Limerick, PA, USA, Catalog No. 200-301-A28; 1:1000 dilution), and ImageJ software was utilized to analyze data.

Western blotting was performed using standard methods. Briefly, cells were washed with cold PBS and lysed in RIPA buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100] containing 1 mM sodium orthovanadate, phosphatase inhibitor cocktail, and protease inhibitor cocktail. Lysates were centrifuged at 16,000 × g for 15 min at 4°C. The protein concentration was measured in a BCA protein assay system (Pierce, Rockford, IL, USA). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes (Whatman, Clifton, NJ, USA), and probed with anti-TonEBP (26 (link)), anti-HO-1, anti-HO-2, anti-p65, anti-lamin B (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Nrf2 (Abcam, Cambridge, UK), and anti-Hsc70 (Rockland, Gilbertsville, PA, USA) antibodies.