Anti rhogdiα

Western blotting was performed according to the standard procedures. Antibodies used were as followed: anti-Nestin (1:2500, Millipore), anti-CD133 (1:200, Miltenyi), anti-SOX2 (1:500, Abcam), anti-Oct4 (1:500, Abcam), anti-Bmi1 (1:1000, Cell Signaling), anti-RhoGDIα (1:1000, Santa Cruz), anti-RhoA (1:1000, Cell Signaling), anti-p-MYPT1 (1:200, Cell Signaling), anti-ROCK1 (1:1000, Santa Cruz).

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.