U 100 insulin

Mice were fasted for 6 hours and, subsequently, injected intraperitoneally with a sterile 2 g/kg glucose solution (50% glucose in 0,9% NaCl, Merck, Germany) using sterile insulin syringes (U-100 Insulin, Terumo, Japan). Blood was drawn from the tail vein immediately before and 15, 30, 60, 120, 150 and 180 minutes after glucose injection. Blood glucose levels were determined using an Accu-Check Aviva glucose analyzer (Roche Diagnostics, Germany).

JKT-1 cells were grown in 10-cm dishes at a density of 4.9 × 10⁶ cells per dish. After 48 h, the JKT-1 cells were scraped in 5 mL cold PBS, pelleted and homogenized in 250 μL cold SI buffer (250 mM sucrose, 3 mM imidazole, pH 7.4, 1 mM PMSF protease inhibitor). Cells were lysed by passing 40 times through a 25G needle (U-100 Insulin, Terumo^®, Somerset, NJ, USA). Nuclei were removed by centrifugation for 10 min at 10,000 g at 4 °C. Protein concentration of the post-nuclear supernatants (PNS) was normalized. PNS were centrifuged for 1 h at 100,000 g at 4 °C. Supernatants correspond to the cytosolic fraction. Pellets, homogenized in an equal volume of SI buffer, correspond to membranes.
Equal amounts (30 μL) of each fraction were resolved on a 12% SDS-polyacrylamide gel. The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon P; Millipore™, Billerica, MA, USA), probed with anti-GPER Ab (Santa Cruz Biotechnology^®, Santa Cruz, CA, USA) and with anti-Rho-GDIα (Santa Cruz Biotechnology^®, Santa Cruz, CA, USA) and anti-transferrin receptor (Invitrogen^®) Abs to control fractionation, then detected using HRP-linked secondary Ab and the ECL System (GE Healthcare^®, Chalfont St. Giles, UK). All experiments were performed in triplicate and the blots shown are representative.