Renal Protein Expression Analysis

As described previously [16 (link)], kidneys were homogenized in a fivefold volume of 20 mmol/L Tris buffer with proteinase inhibitors. After centrifugation at 5,000g for 15 min, the supernatants were centrifuged at 48,000g for 60 min at 4 °C to obtain the cytosolic fraction and membrane fraction. The 50 µg of renal proteins were applied to a 4–20% gradient gel and electroblotted onto polyvinylidene fluoride membranes. The specific protein bands were identified using rabbit polyclonal antibodies for sodium glucose co-transporter 2 (SGLT2) (Abcam, Tokyo, Japan), G6Pase (Abcam), or PEPCK (Abcam) at 1:500 dilution followed by a horseradish peroxidase-conjugated secondary antibody against rabbit immunoglobulin G (Dako, Glostrup, Denmark) and the bands were visualized with diaminobenzidine reaction. The band stained with anti-beta-actin antibody (Abcam) was used as a loading control. The density of the bands was analyzed using the National Institutes of Health Image software program (version 1.63).