Immunoblot Analysis of GLP-1 Protein

Immunoblot analysis was performed as described previously^{48 (link),51 (link)}. Briefly, total protein extract was performed from kidney tissues with sodium dodecyl sulfate-containing sample buffer. The protein concentration of each sample was measured using a Detergent Compatible Protein Assay Kit (Bio-Rad, Japan). Equal amounts of protein extract were fractionated on a 5–20% polyacrylamide gel (ATTO, Japan). The gel was then transferred to a polyvinylidene difluoride (PVDF) membrane using an iBlot Dry Blotting System (Invitrogen, USA). Membranes were blocked for 1 hour at room temperature with phosphate-buffered saline containing 5% skim milk powder, and were then probed overnight at 4 °C with a specific primary antibody to GLP-1(Abcam, Japan). Membranes were washed and further incubated with secondary antibodies for 15 minutes at room temperature. The sites of the antibody–antigen reaction were visualized by enhanced chemiluminescence substrate (GE Healthcare, Japan). Images were analyzed quantitatively using a Fuji LAS-3000 image analyzer (Fuji Film, Japan).