G lisa rhoa activation assay biochem kit

RhoA activities were determined using G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton) according to the manufacturer’s instructions. Briefly, 4 × 10⁵ mature BMDCs were lysed in 70 µl RIPA buffer, and protein concentration was determined using the Precision Red Advanced Protein Assay Reagent (Cytoskeleton). Respective samples were treated with 300 nM nocodazole for 15 min before lysis. All samples were adjusted to a final protein concentration of 0.5 mg/ml. Luminescence signals were measured using a microplate photometer at 600 nm. Wells containing lysis buffer only were used as reference blanks in all experiments.

GTP-bound fraction of RhoA was measured, using the G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton, Inc., TebuBio, Offenbach, Germany). HUASMCs were cultured in 6-well plates and, subsequently, transfected with either control siRNA or RGS5-targeting siRNA to deplete RGS5 Cells, were lysed, and the protein concentration was determined and adjusted for further analyses according to the manufacturer’s instructions. GTP-bound RhoA was normalized to the total RhoA content of the samples, which was assessed by Western blot analysis.

RhoA activity was measured using the G-LISA RhoA Activation Assay Biochem Kit as per the manufacturer's instructions (Cytoskeleton, Denver, CO, USA). Briefly, cells that were seeded and placed in serum-reduced conditions in 175 cm² tissue cell culture flasks were treated with or without Y-27632, rapamycin or doxycycline alone or in combination as indicated for 1 min. (as assessed by time-course experiments to exhibit maximum RhoA expression levels). The cells were then lysed and the activity of RhoA was measured using the G-LISA assay at an absorbance of 490 nm with the limits of detection at 0.05 ng. Following the manufacturer's instructions, levels of active RhoA were then normalised to total Rho (-A, -B, -C) levels as measured using immunoblotting (as detailed above). Mouse monoclonal anti-Rho (-A, -B, -C) primary antibody (diluted 1:2000; Millipore) and goat polyclonal antimouse immunoglobulin G-HRP conjugated secondary antibody (diluted 1:2000; Dako) were used.

Rho-associated kinase (ROCK) activity in colon tissue was detected by ROCK activity assay kit (CSA001, Millilpore, MA, US). The active and total RhoA in colon tissue was detected by G-LISA RhoA activation assay biochem kit (BK124, Cytoskeleton, Inc., CO, US), and total RhoA ELISA kit (BK150, Cytoskeleton, Inc., CO, US), respectively. The colon tissue sample was prepared according to the instruction of these commercial kits.

Active RhoA was measured by G-LISA RhoA Activation Assay Biochem kit (colorimetric assay, Cytoskeleton, USA) following the manufacturer’s instruction. And the signal was measured at 490 nm with a microplate reader (MRX, Dynatech Laboratories). Total RhoA protein expression of each group was measured by WB, as described above. Results were expressed as fold activity of stimulated in relation to non-stimulated controls normalized to total RhoA protein content.