Isolated CD4+CD28+ and CD4+CD28null T lymphocytes from eight patients were pooled and cultured in serum-free conditions for 1 h. After this, lymphocytes were cultured in completed medium alone or in the presence of IL-15 stimulation (50 ng/mL) for 5 min. It was well documented that the GTPases activation occurs in a very short period of time (a few minutes), and as it is said in the manufacturer instructions of G-LISA activation assays (Cytoskeleton, Inc., Denver, CO, USA), Rho proteins are generally activated very rapidly and transiently (30 s to 30 min). Following this, we decided to incubate under IL-15 stimulation for 5 min. After the incubation, cold PBS was added to the cells in order to stop the stimulation. Protein extracts were obtained from each condition and both basal levels of RhoA and activated forms of RhoA, Rac1, and Cdc42 were measured following the manufacturer instructions using G-LISA activation assays (Cytoskeleton, Inc., Denver, CO, USA). Calpeptine was used as the positive control for RhoA activation.
G lisa activation assay
Manufactured by Cytoskeleton
Sourced in United States
The G-LISA Activation Assay is a laboratory equipment product designed to quantify the activation state of specific proteins. It utilizes a proprietary technique to measure the active, GTP-bound form of target proteins. The assay provides a reliable and efficient method for researchers to analyze and study the activation dynamics of these proteins within their experimental systems.
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18 protocols using g lisa activation assay
1
Rapid Rho GTPase Activation Profiling
2
Rho and Rac GTPase Activation Assay
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RhoA- and Rac-GTP levels were detected using colorimetric GLISA activation assays (Cytoskeleton, Denver, CO), according to manufacturer's protocol. Signal produced by the detection reagent, proportional to the amount of Rho- or Rac-GTP, was detected by measuring absorbance at 490nm using a Synergy 2 plate reader (BioTek, Winooski, VT). Constitutively active Rho and Rac were used as positive controls.
3
Osteogenic Differentiation of Arhgap21+/- BM Cells
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Arhgap21+/− and WT BM cells were induced for osteogenic differentiation as described above and total protein was extracted on days 0, 7 and 16. Three pools of BM cells containing three mice each were evaluated per group. Day 0 denotes the addition of mineralizing media. RhoA and Cdc42 activities were determined in protein extracts using G-Lisa Activation Assays (Cytoskeleton, Inc.).
4
Rho and Rac GTPase Activation Assay
5
Measurement of Rho GTPase Activation
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Cells were seeded in six-well plates (5 × 105 cells per well) and maintained for 12 h in a CO2 incubator. To measure Rac-1, RhoA and CDC42 GTPases, they were treated with 10 µM NT4 in DMEM-0.1% BSA, serum-free, for 12 h at 37 °C and then incubated with 0.1 μg/mL hEGF (Cell Signaling) for 2 min for Rac-1 and CDC42 GTPases and for 5 min for RhoA GTPase. Cells were washed with cold PBS, lysed and processed for colorimetric G-LISA activation assays (Cytoskeleton, Denver, CO, USA), according to the manufacturer’s instructions. The activated forms of the G proteins were detected by incubation with specific primary antibody, followed by a secondary antibody conjugated with HRP and a detection reagent. The signal was read by measuring absorbance at 490 nm using a microplate reader. The experiment was repeated three times.
6
Measuring Small G-Protein Activity
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Cells were treated with scr siRNA or siSmn and the activity of small G-proteins (Rac1, RhoA and Cdc42) was measured by utilizing G-LISA Activation Assays (BK135, Cytoskeleton, Denver, Colorado, USA). The assays were performed according to the manufacturer’s instructions. Shortly, cell lysate was added to wells linked with the binding domain of an effector molecule of the small GTPases. The active protein was bound to the well bottom and was subsequently detected via specific small G-protein antibodies. An HRP-linked secondary antibody allowed colorimetric determination of the degree of activation.
7
Rac1, RhoA, and Cdc42 GTPase Activation Assays
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G-LISA Activation Assays (Cytoskeleton, Inc., Denver, CO, USA) were used to compare the levels of activated Rac1 (BK128), RhoA (BK124), and Cdc42 (BK127) GTPases in different groups of cells after LPA and PMA stimulations. Lysates were prepared according to the protocol, except for lysates for G-LISA Cdc42 Activation Assay, in which lysis buffer was a mixture of GL35:GL36 buffers in ratio 1:1. This was done upon an advice of technical support of Cytoskeleton, Inc. The Precision Red Protein Assay reagent was used for quantification of protein concentration in lysates, which was around 0.5 mg/mL. Active proteins were detected by incubation with primary antibody detecting given GTPase and appropriate secondary antibody conjugated with horseradish peroxidase (HRP). Colorimetric signal was measured at 490 nm using a microplate spectrophotometer. The data were presented as ratio between stimulated and nonstimulated (starved for 24 h) cells with normalization to the control cells.
8
Measuring Small GTPase Activity in iPSC-derived MGE Progenitors
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Activity of small GTPases (CDC42, Rac1, and RhoA) in the MGE progenitors derived from the null and direct iPSC lines was measured by G-LISA Activation Assay (Cytoskeleton Inc.) according to the manufacturer's protocol. The MGE progenitors were starved overnight, collected by centrifugation (300 × g, 4 min, RT), plated on 0.03% and/or 0.1% hydrogels, and incubated at 37 °C for 1 h. Afterward, the cells were lysed with an appropriate lysis buffer and centrifugated (10,000 × g, 1 min, 4 °C). Supernatants were immediately frozen and kept at −80 °C until the G-LISA Activity Assay. Protein concentration was measured by Precision Red™ Advanced Protein Assay (Cytoskeleton Inc.).
9
Rac1 GTPase Activity in BMDCs
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BMDCs from Sema3e−/− and WT mice were first stimulated with CCL21 (20 ng/ml) for 0, 0.5, 1, 5, 15 and 30 min. Then, GTPase activity of Rac1 was measured in snap-frozen BMDC extracts by G-LISA activation assay according to the manufacturer’s instructions (Cytoskeleton, Denver, CO).
10
Quantifying Rho GTPase Activity in Glomeruli
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The activity of RhoA, Rac1 and Cdc42 in isolated glomeruli was measured by a G-LISA activation assay following the manufacturer’s instructions (Cytoskeleton, Inc., Denver, CO). Briefly, isolated glomeruli were immediately lysed using the ice-cold cell lysis buffer provided with the G-LISA kit, and magnetic beads and cell debris were removed by centrifugation at 3,000 × g for 15 min at 4 °C. Equal amounts of proteins (10 to 50 μg) were applied to the Rho-GTP affinity G-LISA plate and incubated for 30 min at room temperature. The active GTP-bound forms of RhoA, Rac1 and Cdc42 were measured using indirect immunodetection, followed by a colorimetric reaction at 490 nm on a microplate spectrophotometer.
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