Gst rhotekin rbd fusion protein

Pull-down assay was used to assess RhoC activity as previously described (22 (link)). For these experiments, A549 cells were harvested with RIPA lysis buffer coupled with a protease inhibitor, centrifuged at 13,000 × g at 4°C for 20 min to obtain a supernatant. The extracts were incubated for 40 min at 4°C with 20 µg glutathione beads bound to GST-Rhotekin RBD fusion protein (Cytoskeleton, Inc., Denver, CO, USA), then washed 3 times with washing buffer. The Rho content of the beads was eluted and boiled to denaturation, and then separated using 15% SDS-PAGE, transferred to a PVDF membrane and immunoblotted with a RhoC antibody.

Active RhoA (GTP-bound) was captured using the Rho Activation Assay Biochem Kit (Cytoskeleton, Inc., Japan). PANC-1 cells that were grown in logarithmic phase in a 10-cm plate were starved in serum-free medium containing Y-27632/Rhosin for 24 h and were subsequently stimulated with gastrin. Cells were lysed in a buffer containing 100 mM NaCl, 20 mM Tris-HCL, 1% Triton X-100, 2 mM NaF, and protease inhibitors (Roche Diagnostics). Cell extracts were homogenized and incubated overnight with GST-rhotekin-RBD fusion protein (Cytoskeleton, Japan). Active GTP-RhoA in the lysates was evaluated by immunoblotting with a monoclonal RhoA antibody.