Gst rhotekin rbd

Antibodies to RhoA, S6K, TSC2, p190RhoGAP, TIAM1, IQGAP, HA-Tag, and Myc-Tag were purchased from Santa Cruz and antibodies to actin, Flag, and Phospho-70S6K were from Life Sciences. Anti-ANDV Gn monoclonal antibody was purchased from United States Biologicals. Anti-N-protein polyclonal rabbit sera made to NY-1V N protein was previously described (25 (link), 26 (link)), and RhoA-glutathione S-transferase (GST) activation assays were performed with GST-Rhotekin-RBD from Cytoskeleton Inc. Bradykinin was purchased from Sigma, and fasudil and Y27632 were purchased from Selleck Chemicals.

The capacity of RhoA–GTP to bind to GST–rhotekin–RBD was used to determine the cellular levels of active GTPase. Cells were lysed in extraction buffer (50 mM Tris.HCl pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 1% NP-40, 10% glycerol, 1 mM DTT, protease and phosphatase inhibitor cocktail) and the RhoA pull-down assay was performed by incubation of cleared lysates with GST–rhotekin–RBD (Cytoskeleton Inc) for 45 min at 4 °C, followed by four washes of the beads in wash buffer (50 mM Tris.HCl, pH 7.6, 150 mM NaCl, 10 mM MgCl_2, 1 mM DTT, protease and phosphatase inhibitor cocktail). Bound proteins were solubilized by the addition of 25 μl of SDS–polyacrylamide gel electrophoresis (SDS–PAGE) Laemmli loading buffer, followed by separation on 12.5% SDS–PAGE gels and western blotting for RhoA.
MEF focus formation assay. Primary Pik3ca^H1047R+neo;Flpe-ER^T2 MEFs (passage 2) were seeded at 10⁶ cells per 6 cm dish in standard culture medium in the presence of 4-OHT with or without Y27632 (10 μM) or H1152 (0.5 μM) until they reached a confluent monolayer. Medium was replaced every 2 days, containing fresh inhibitors, and confluent monolayer cultures stained with crystal violet after 10 days of total culture to reveal the presence of transformed foci.

These experiments were performed as described previously^{16 (link)}. GST-Rac1 (Addgene, #12200) and GST-RhoA (Addgene, #12202) were purified from the BL21 (DE3) E. coli strain, and GST-PAK1 CRIB and GST-rhotekin RBD domains were purchased from Cytoskeleton, Inc. Anti-FLAG, anti-Myc, anti-p-ERK, anti-p-JNK, anti-ERK (Cell Signaling Technology, Danvers, MA, USA), anti-Rac1, anti-Cdc42 (BD Transduction Laboratories), anti-RhoA (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-GFP (Invitrogen) were purchased from the indicated commercial sources. The anti-PLCE1 antibody has been previously described by Hinkes et al.^{8 (link)}. Coimmunoprecipitation was performed using EZview Red Anti-FLAG M2 or Anti-c-Myc Affinity Gels (Sigma-Aldrich, St.Louis, MO, USA). The intensities of immunoblots were analyzed using ImageJ.