Rhoa activation assay biochem kit

Active GTP-bound RhoA can be presented by RhoA activity assay. Rho activation pull-down assay was performed in the lysates collected from lung tissue using a RhoA activation assay Biochem kit (Cytoskeleton Inc., Denver, CO, USA) according to manufacturer's indications. Briefly, supernatants were incubated with Rhotekin Rho-binding domain at 4°C (×1 h) on a rotator. Bead-precipitated proteins were fractionated and immunoblotted with antibody against RhoA.

Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany). All test substances were diluted in Dulbecco´s modified eagle medium nutrient mixture F-12 (DMEM/F12, Gibco-Invitrogen, Karlsruhe, Germany) containing 10% fetal bovine serum (Gibco-Invitrogen), 1% Penicillin and Streptomycin (Gibco-Invitrogen) and 10 μM retinoic acid. The non-steroidal cyclooxygenase inhibitor Ibuprofen, the ROCK inhibitors Y-27632 and Fasudil, and the cAMP analogue 8-Br-cAMP (8-Bromoadenosine 3′, 5′-cyclic monophosphate) were purchased from Sigma-Aldrich. The RhoA Activation Assay Biochem Kit (bead pull-down format) was purchased from Cytoskeleton Inc. (Denver, CO, USA). Alamar Blue cell viability assay to measure cytotoxic effect was purchased from Trinova (Giessen, Germany).

RhoA activation was assessed by loading the RhoA protein in cell lysates, using the RhoA Activation Assay Biochem Kit (Cytoskeleton, Denver, CO, USA), based on the method described by Cao et al. [25 (link)], whereby cellular GTP-bound RhoA is detected by Western blot after affinity precipitation with a fusion protein containing glutathione-S-transferase (GST) and the Rho-binding domain of Rhotekin (GST-RBD). Briefly, total cellular proteins were extracted and quantitated with a Bradford protein assay (Bio-Rad, Hercules, CA, USA). These proteins were then incubated with 25 μg aliquots of brightly colored glutathione affinity beads coupled to GST-RBD in order to pull down the GTP-bound form of RhoA. RhoA activation was quantitatively analyzed by standard Western blot analysis using anti-RhoA antibody.

RhoA activity was assessed using a RhoA Activation Assay Biochem Kit according to the manufacturer’s instructions (Cytoskeleton; cat. # BK036). Briefly, GTP-RhoA was immunoprecipitated from whole-cell lysates with glutathione S-transferase–tagged Rhotekin bound to glutathione agarose beads. The beads were washed, and immunoprecipitates were analyzed by WB using a RhoA-specific monoclonal antibody. The lysate was also probed for total RhoA, and GTP-RhoA was normalized to total RhoA levels.

Western blotting was performed using standard methods as previously reported [19 (link)]. Antiprofilin-1 antibody (#3237), anti-ROCK1 (#4035), and anti-p-eNOS antibodies (#9570) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin antibody (sc-47778) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
RhoA activation was assessed using the RhoA Activation Assay Biochem Kit (Cytoskeleton, Denver, CO, USA) according to previously described methods [18 (link)].

To measure the level of RhoA‐GTP (GTP‐bound state, active form of RhoA) in the bEnd.3 cells, mouse brain capillaries, and capillary‐depleted brain samples, RhoA activation assay biochem kit (Cytoskeleton, Denver, CO, USA) was used in accordance with the manufacturer's guidelines. In brief, the samples were lysed with cell lysis buffer (RIPA buffer) and incubated with predetermined amount of Rhotekin‐RBD beads (bind specifically to the GTP‐bound form of Rho) for 1 h at 4 °C. Next, the samples were centrifuged at 5000 g at 4 °C for 1 min, and the bead pellets were washed with wash buffer. After centrifugation, again the supernatant was removed, and remaining bead pellets were boiled with 10 μL of 2× laemmli sample buffer for 5 min at 85 °C. The samples were loaded on the 4–12% Nupage bis‐tris gels (Thermo Fisher Scientific). Anti‐RhoA antibody was used for immunoblotting.