The culture medium of HEK293T cells expressing rDA1h or DA2h was replaced by 100 μl Tyrode’s solution before experiments. For the group with GTPγS treatment, cells were subsequently incubated with 50 μg/ml digitonin (Sigma-Aldrich) for 5 min to permeabilize the cell membrane and washed for 2 times with 100 μl Tyrode’s solution. The cells were then incubated with Tyrode’s solution containing 100 μM GTPγS (Sigma-Aldrich) for 10 min. Various concentration of DA (ranging from 0.01 nM to 100 μM) was applied. The fluorescence signals were measured using Opera Phenix high-content screening system (PerkinElmer) mentioned above.
Gtpγs
Manufactured by Merck Group
Sourced in United States, Hungary, Germany
GTPγS is a non-hydrolyzable analog of guanosine-5'-triphosphate (GTP). It is commonly used as a research tool in biochemical and cell biology studies to investigate the role of G-proteins and their associated signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
35s gtpγs, by PerkinElmer (11 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Digitonin, by Merck Group (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
3h sch23390, by PerkinElmer (3 mentions)
Tris hcl, by Merck Group (3 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (3 mentions)
Opera phenix high content screening system, by PerkinElmer (3 mentions)
3h damgo, by PerkinElmer (3 mentions)
U69 593, by Merck Group (3 mentions)
8 oh dpat, by Merck Group (2 mentions)
Unlabeled gtpγs, by Merck Group (2 mentions)
35s gtpγs 1250 ci mmol, by PerkinElmer (2 mentions)
Bac to bac baculovirus expression system, by Thermo Fisher Scientific (2 mentions)
Gdc0068, by Abcam (2 mentions)
Integrin α5 peptides, by GenScript (2 mentions)
Alexa 546 conjugated transferrin, by Thermo Fisher Scientific (2 mentions)
Hygromycin, by Merck Group (2 mentions)
Fluphenazine, by Merck Group (2 mentions)
Tetracycline, by Merck Group (2 mentions)
57 protocols using gtpγs
1
Receptor Activation in HEK293T Cells
2
Measurement of Gt Activation by Opsin Membranes
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Gt was extracted and purified from frozen bovine ROS membranes as described in Preparation of Opsin Membranes. The intrinsic increase in the fluorescence from Gtα was measured with a L55 luminescence spectrophotometer (PerkinElmer Life Sciences) using an excitation and emission wavelengths of 300 and 345 nm, respectively39 (link),40 (link). To test the effect of YC-001 on the basal activity of rod opsin, opsin membranes were incubated for 15 min at 20 °C with a 40 µM concentration of either YC-001, the non-active analog-YC-014, 9-cis-retinal or a co-treatment with 9-cis-retinal and YC-001. DMSO-treated opsin membranes were used to obtain a baseline for the basal activity of opsin. The molar ratio of opsin to Gt was 1:10, with opsin at a concentration of 100 nM and Gt at 1000 nM. Opsin membranes treated with either 9-cis-retinal alone or a co-treatment of 9-cis-retinal and YC-001 were bleached for 1 min with a fiber light source (Dolan Jenner Industries Inc., Boxborough, MA) equipped with a 480 to 520 nm band-pass wavelength filter (Chroma Technology Corporation, Bellows Falls, VT, USA). This step was followed by the addition of 300 μM GTPγS (Sigma-Aldrich) to determine the GTPγS-induced complex dissociation and corresponding fluorescence changes. Gt activation rates were determined and plotted for the first 100 s of the Gt activation assay40 (link),67 (link).
3
Quantifying Rhodopsin-mediated G Protein Activation
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The intrinsic fluorescence increase from Gαt was measured with a L55 luminescence spectrophotometer (PerkinElmer Life Sciences) operating at excitation and emission wavelengths of 300 and 335 nm, respectively23 (link),59 (link). The ratio of Gt to Rh was 20:1, with Gt at a concentration of 1000 nM and Rh at 50 nM. Gt was pre-incubated with a two molar excess of either Nb5 or Nb17 to determine their effects on Rh*-mediated Gt activation rates. This was followed by the addition of 300 µM GTPγS (Sigma-Aldrich) to determine the GTPγS-induced complex dissociation and fluorescence changes58 (link). Samples were bleached for 1 min with a Fiber-Light delivered through a 480–520 nm long pass wavelength filter prior to the fluorescence measurements. Gt activation rates were determined for the first 100 s in a Gt activation assay58 (link).
4
GST Pull-down Analysis of Rab25-Integrin Interaction
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GST pull-downs were performed as described previously (Caswell et al., 2007 (link)). Briefly, His–Rab25 was expressed in Escherichia coli strain BL-21 and purified by sequential Ni-affinity and size-exclusion chromatography. Rab25 was loaded with GTPγS by incubation in PBS containing 4.3 mM EDTA and 0.11 mM GTPγS (Sigma-Aldrich) at room temperature for 1 h, before addition of MgCl2 to 6.5 mM. Glutathione–agarose beads (Thermo Fisher Scientific) were bound to bacterially expressed purified GST–β1 integrin cytoplasmic tail truncations, washed extensively and incubated with His–Rab25 GTPγS for 2 h at 4°C. After extensive washing to remove unbound proteins, bound proteins were eluted by boiling in Laemmli sample buffer and proteins resolved by SDS-PAGE, followed by Coomassie Blue staining or western blotting.
5
Purification and Pull-Down of GTPase-Binding Proteins
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GST-hRaf-RBD (1-147), GST-hRhotekin-RBD (7-89) and GST-hGGA3-VHS-GAT (1-313) were expressed in E. coli and purified by batch method on GSH-agarose (ThermoFisher Scientific). Per pull-down, we used 80 μg of fusion protein. GST-Pak-PBD beads were obtained from Cytoskeleton; per pull-down, 50 μg of fusion protein were used. As a positive control, we used GTPγS-loaded HeLa extract (see below) prepared by sequentially adjusting the cleared lysate to 15 mM EDTA, adding to 0.2 mM GTPγS (Sigma-Aldrich) and incubating at 30°C for 15 min, transferring to ice and adjusting to 60 mM MgCl2.
SiRNA-treated HeLa cells (one 15 cm dish per pull-down sample) were washed once with ice-cold PBS and collected in GTPase lysis buffer (G-LB: 50 mM Tris pH 7.2, 500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 1% phosphatase inhibitor cocktail [Calbiochem] + one tablet complete mini protease inhibitor, EDTA-free [Merck] per 7 mL). Crude lysates were immediately centrifuged at 20,800× g at 4°C for 2 min and cleared lysates incubated with GST-hRaf-RBD, GST-hRhotekin-RBD or GST-Pak-PBD beads rotating at 4°C for 1 h. Unbound supernatant was discarded, beads were sucked dry using a needle and washed once in 25 mM Tris pH 7.2, 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100. Bound proteins were eluted using 1× Laemmli sample buffer.
SiRNA-treated HeLa cells (one 15 cm dish per pull-down sample) were washed once with ice-cold PBS and collected in GTPase lysis buffer (G-LB: 50 mM Tris pH 7.2, 500 mM NaCl, 10 mM MgCl2, 1% Triton X-100, 1% phosphatase inhibitor cocktail [Calbiochem] + one tablet complete mini protease inhibitor, EDTA-free [Merck] per 7 mL). Crude lysates were immediately centrifuged at 20,800× g at 4°C for 2 min and cleared lysates incubated with GST-hRaf-RBD, GST-hRhotekin-RBD or GST-Pak-PBD beads rotating at 4°C for 1 h. Unbound supernatant was discarded, beads were sucked dry using a needle and washed once in 25 mM Tris pH 7.2, 150 mM NaCl, 10 mM MgCl2, 1% Triton X-100. Bound proteins were eluted using 1× Laemmli sample buffer.
6
Receptor Activation in HEK293T Cells
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The culture medium of HEK293T cells expressing rDA1h or DA2h was replaced by 100 μl Tyrode’s solution before experiments. For the group with GTPγS treatment, cells were subsequently incubated with 50 μg/ml digitonin (Sigma-Aldrich) for 5 min to permeabilize the cell membrane and washed for 2 times with 100 μl Tyrode’s solution. The cells were then incubated with Tyrode’s solution containing 100 μM GTPγS (Sigma-Aldrich) for 10 min. Various concentration of DA (ranging from 0.01 nM to 100 μM) was applied. The fluorescence signals were measured using Opera Phenix high-content screening system (PerkinElmer) mentioned above.
7
Assay for Active Daam1 in Cells
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For the detection of active Daam1, GST-RhoA beads were incubated with 0.1 mmol/l GTPγS (Sigma) at 30°C for 15 min with constant agitation. Equal volumes of total cellular protein were incubated with GST-RhoA beads captured on MagneGST Glutathione Particles (Promega, Madison, WI, USA) at 4°C with constant rotation for 90 min. The beads were washed three times with washing buffer (4.2 mmol/l Na2HPO4, 2 mmol/l KH2PO4, 140 mmol/l NaCl and 10 mmol/l KCl, pH 7.2). At the end of this period, the beads were captured using a magnet on a magnetic stand. After being washed three times with ice-cold buffer, the beads were resuspended in Laemmli buffer, boiled, and subjected to western blot analysis. SDS-PAGE and western blotting were performed using standard methods.
8
Assay of Diverse Pharmacological Agents
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Quinpirole, buspirone, 8-OH-DPAT, SCH 23390, [35S]GTPγS and GTPγS were purchased from Sigma (St. Louis, MO, USA). SKF 38393 was purchased from Tocris (Bristol, United Kingdom). The cAMP Assay Kit was purchased from CISBIO (Catalog Number: 62AM4PEC). WAY100635 was obtained from Selleck (Texas, USA) and was dissolved in 0.9% saline. l-THBr was synthesized by the Department of Complex Prescription of Traditional Chinese Medicine (TCM), China Pharmaceutical University. It was dissolved in 0.1 mol/L H2SO4, diluted with sterile water and adjusted to pH 5–6 with 0.1 mol/L NaOH. The vehicle was prepared as above without drug. Diazepam (DZP) was purchased from Tianjin Jinyao Amino Acid Co., Ltd. (Tianjin, China) and was dissolved with control vehicle. For in vitro assays, all compounds were dissolved in DMSO and diluted with a solution of HBSS plus 20 mM HEPES.
9
Affinity Capture of Rho GTPases
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When bound to GTP, Rho GTPases interact with their effectors (Aspenström, 1999 (link)). PAK1 interacts with Cdc42·GTP and Rac1·GTP through its p21 binding domain (PBD). Replicate aliquots of the HeLa clones with regulatable expression of HA-Dock10 cultured for 24 h in the presence and absence of dox were assayed by pull down using GST-PAK1-PBD bound beads. Cell were lysed in Lysis Buffer B containing 50 mM Tris⋅Cl pH 7.2, 0.5 M NaCl, 10 mM MgCl2, 1% Triton X-100, and cOmplete EDTA free. GST-PAK1-PBD beads (40 µl) were added to cleared lysates (500 µl) and incubated with end-over-end shaking at 4°C for 1 h. Beads were then washed in Tris Wash Buffer B containing 50 mM Tris-Cl pH 7.2, 150 mM NaCl, 10 mM MgCl2, 0.5% Triton X-100, and cOmplete EDTA free, and SDS loading buffer was added. Proteins were denatured for 10 min at 100°C, loaded onto SDS-PAGE gels and immunoblotted using Cdc42 or Rac1 specific antibodies. In different control experiments, HeLa cells were treated with 100 ng/ml epidermal growth factor (EGF, Sigma-Aldrich), or HeLa protein extracts were incubated either with 1 mM GDP or 100 µM GTPγS (Sigma-Aldrich), a non hydrolyzable GTP analog.
10
Measuring CB2 Receptor Activation via GTPγS Binding
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