Rhoa 67b9

Cells were lysed for 15 min at ice bath using a lysis buffer (1% Triton X-100, 20 mM Tris pH 7.5, 1 mM MgCl₂, 150 mM NaCl, 1 mM Na₃VO₄, 50 mM NaF, 1.5 mM EDTA, 10% glycerol, 20 mM β-glycerophosphate, 10 µg/ml aprotonin, 1 µM pepstatin A) containing 5 µM PUGNAc (an OGA inhibitor; Toronto Research Chemicals, Inc., North York, Canada). Protein samples (50 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA, Darmstadt, Germany). The membrane was blocked with 5% non-fat dried milk in TBST for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. Antibodies specific to O-GlcNAcylation (RL2; 1:1,000; Affinity BioReagents, Golden, CO, USA) and OGT (F-12; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were used. RhoA (67B9; 1:1,000), MLC (3672; 1:1,000) and phosphorylated (p-)MLC (3671; 1:1,000) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). GAPDH antibody (sc-25778; 1:2,000) and horseradish peroxidase-linked goat anti-mouse (sc-2005; 1:2,000) and goat anti-rabbit (sc-2004; 1:2,000) IgG secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Development was carried out using an enhanced chemiluminescence western blotting detection reagent (GE Healthcare Life Sciences, Chalfont, UK).