Rhoa pull down activation assay biochem kit

Chang liver and T-REx-HeLa were purchased from ATCC (#CCL-13), and Invitrogen (R714-07), respectively. G418 sulfate was purchased from AG Scientific, doxycycline from BD Biosciences, and anti-MYPT1, anti-pMYPT1, anti-RhoA antibody were purchased from Cell Signaling. RhoA Pull-down Activation Assay Biochem kit was obtained from Cytoskeleton, and Alexa Fluor 488 goat anti-mouse IgG antibody, blasticidin, pcDNA6/TR vector and TRIZOL reagent were purchased from Invitrogen. Rhodamine phalloidin was obtained from Life Technologies, pSuperior.neo vector from OligoEngine, and Mo-MuLV reverse transcriptase from Promega. In Situ Cell Death Detection Kit was purchased from Roche. Annexin V-FITC Apoptosis Detection Kit, anti-α-tubulin antibody, blebbistatin, cycloheximide, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), HRP-conjugated goat anti-rabbit IgG antibody, HRP-conjugated goat anti-mouse IgG antibody, human tumor necrosis factor-α (hTNF-α), phosphatase inhibitor cocktail II, III, propidium iodide, protease inhibitor mixture, RNase A and Y-27632 were purchased from Sigma. DNAs were synthesized from Cosmogenetech (Korea). The His-tagged Tat-C3 transferase exoenzyme (pHis-Tat-C3) expression vector was provided by Jae Bong Park and the recombinant C3 transferase was prepared as previously described [20 (link)].

HN12 cells and Cal27 cells were cultured to 50% confluency, and then serum starved overnight. To induce RhoA activation, cells were treated with 5 μM LPA, 1 μM Ang II, or 10 μM CP-55940 for 5 min. Active RhoA levels were measured using the RhoA Pull-Down Activation Assay Biochem Kit (bead pull-down format; Cytoskeleton) following the manufacturer instruction using a modified lysis buffer (50 mM Tris-HCl (pH 7.2), 500 mM NaCl, 10 mM MgCl₂, 0.1% SDS, 1% NP-40). Briefly, after stimulation, samples were lysed and protein concentrations were quantified using DC Protein Assay (BioRad). Samples were adjusted to the same concentration with lysis buffer and 500 μg of each protein lysate was added to 15 μL GST-tagged Rhotekin-RBD bound to Sepharose beads. Samples were incubated while rocking at 4°C for 1.5 h. Beads were then washed, eluted in Laemmli sample buffer, and analyzed by western blot using a mouse monoclonal anti-RhoA antibody (Cytoskeleton).

Active RhoA levels were measured using the RhoA Pull-down Activation Assay Biochem Kit (BK036-S, Cytoskeleton Inc.) using GST Rhotekin beads. The levels of the GTP-RhoA associated with GST-Rhotekin-RBD were quantified by western blot analysis. Briefly, A293 cells were plated in a 6-well plate at a density of 3.5 x 10⁵ cells per well and transfected with 1 μg Gα_i1, 100 ng c-myc-PRG and, 250 ng RhoA-HA per well using 1:3 DNA: Lipofectamine 2000 ratio. 20 hr after transfection, cells were cultured in serum-free media for 4 hr. Cells in each well were then lysed with 300 μL of RhoA lysis buffer with 1×PI (included with the kit), and lysates were equalized for total protein amount. Samples from two wells were pooled for each experimental group (total 600 μL, ~600 μg protein per experimental group). The lysates were incubated with 50 μg of GST-Rhotekin bound beads in an end-over-end rotator for 1 hr at 4 °C. Beads were washed twice with wash buffer (included with the kit), eluted in 40 μL 1× Laemmli sample buffer, and analyzed by western blot using an anti-HA (1:2000), anti-c-myc antibody (1:2000), and anti-Gα_i1/2 antisera (1:3000). Band intensities were quantified using Image Studio Lite (version 5.2)