active rap1 detection kit  (Cell Signaling Technology Inc)

Journal: JCI insight

Article Title: Epac1 contributes to apremilast-mediated rescue of pemphigus autoantibody-induced loss of keratinocyte adhesion.

doi: 10.1172/jci.insight.187481

Figure Lengend Snippet: Figure 5: Apremilast induces phosphorylation of Pg and Rap1 activation in a

Article Snippet: Rap1 pulldown Rap1 GTPase activation was measured using the Active Rap1 Detection Kit (Cell Signaling, Leiden, Netherlands) following the manufacturer’s instructions.

Techniques: Phospho-proteomics, Activation Assay

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet: (A) Family tree of human superfamily of Ras-like GTPases. (B) Sequence alignment of various Ras-family GTPases. Residues mediating drug resistance to adagrasib are highlighted in light orange (rare) and orange (common). (C) Covalent modification of RalA(G23C) with compounds 1–10 (50 μM, 12 h). (D) Intact protein mass spectra of RalA(G23C)·GDP and RalA(G23C)·GDP·MRTX1257 adduct. (E) Time-dependent covalent modification of RalA(G23C) with different compounds (50 μM). (F) Differential scanning fluorimetry of RalA(G23C)·GDP and RalA(G23C)·GDP·divarasib adduct. (G) Covalent modification of Rap1A(G12C) with compounds 1–10 (50 μM, 12 h). (H) Intact protein mass spectra of Rap1A(G12C, L96F)·GDP and Rap1A(G12C,L96F)·GDP·divarasib adduct. (I) Time-dependent covalent modification of Rap1A(G12C) and Rap1A(G12C, L96F) with different compounds (50 μM). (J) Differential scanning fluorimetry of Rap1A(G12C, L96F)·GDP and Rap1A(G12C, L96F)·GDP·divarasib adduct. See also .

Article Snippet: Rap1A activity was measured with the Active Rap1A Detection Kit from Cell Signaling Technology (CST) 8818, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Sequencing, Modification

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet: (A) Peptides showing significant differences in HDX at any time point (>0.35 Da and >4.5%) mapped onto a homology model of RalA based on adagrasib-bound K-Ras(G12C) (PDB: 6USZ) according to the legend. (B) Intrinsic or RAPGEF5- or EDTA-mediated nucleotide exchange of BODIPY-GDP with Rap1A(G12C, L96F)·GDP and Rap1A(G12C, L96F)·GDP·divarasib adduct. (C) Immunoblot of HeLa cells transiently overexpressing EGFP-RalA(WT) and EGFP-RalA(G23C). (D) RalA activity measured by RalA G-LISA. HeLa cells were transiently transfected, treated with different concentrations of MRTX1257 for 12 h, and lysates were tested at 0.5 mg/mL. Data are presented as mean ± SEM (n = 2) and are representative of three independent experiments. (E) IP of active GTP-bound Rap1 using GST-RalGDS-RBD of HeLa cells transiently overexpressing EGFP-Rap1A(WT) and EGFP-Rap1A(G12C, L96F) and treated with different concentrations of divarasib. See also .

Article Snippet: Rap1A activity was measured with the Active Rap1A Detection Kit from Cell Signaling Technology (CST) 8818, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Western Blot, Activity Assay, Transfection

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: Rap1A activity was measured with the Active Rap1A Detection Kit from Cell Signaling Technology (CST) 8818, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet: (A) Covalent modification of Rac1(G12C) with compounds 1–10 (50 μM, 12 h). (B) Covalent modification of RhoA(G14C) with compounds 1–10 (50 μM, 12 h). (C) Time-dependent covalent modification of Rac1(G12C), RhoA(G14C), and Rac1(WT) with divarasib (50 μM). (D) Time-dependent covalent modification of various Rac1 mutants with divarasib (50 μM). (E) Covalent modification of Rab1A(S20C) with compounds 1–10 (50 μM, 12 h). (F) Covalent modification of Rab5C(S30C) with compounds 1–10 (50 μM, 12 h). (G) Time-dependent covalent modification of Rab1A(S20C), Ypt1(S17C), Rab5C(S30C), and RabL5(WT) with MRTX1257 (50 μM). (H) Time-dependent covalent modification of various Rab1A mutants with MRTX1257 (50 μM). (I) Peptides showing significant differences in HDX at any time point (>0.35 Da and >4.5%) mapped onto a homology model of Rab1A based on adagrasib-bound K-Ras(G12C) (PDB: 6USZ). (J) Differential scanning fluorimetry of Rac1(G12C, K96H)·GDP and Rac1(G12C, K96H)·GDP·divarasib adduct. (K) Rac1 activity measured by Rac1 G-LISA. HeLa cells were transiently transfected, treated with different concentrations of divarasib for 12 h, and lysates were tested at 0.5 mg/mL. Data are presented as mean ± SEM ( n = 2) and are representative of three independent experiments. See also , , and .

Article Snippet: The effects of Rac1 inhibition on downstream signaling was assessed on COS7 cells via the Active Rac1 Detection Kit from Cell Signaling Technology (CST) 8818S, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Modification, Activity Assay, Transfection

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet: (A and B) Representative binding poses from covalent MD simulations of divarasib, and selected ligands in Rac1(G12C) (A) and Rab5C(S30C) (B), respectively. (C) Chemical structures of novel SII pocket inhibitors to improve targeting of Rab and Rho GTPases. (D) Covalent modification of Rac1(G12C) with compounds 1–22 (50 μM, 1 h). (E) Covalent modification of Rab1A(S20C) with compounds 1–22 (50 μM, 1 h). (F) Covalent modification of Rab5C(S30C) with compounds 1–22 (50 μM, 12 h). See also and .

Article Snippet: The effects of Rac1 inhibition on downstream signaling was assessed on COS7 cells via the Active Rac1 Detection Kit from Cell Signaling Technology (CST) 8818S, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Binding Assay, Modification

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: The effects of Rac1 inhibition on downstream signaling was assessed on COS7 cells via the Active Rac1 Detection Kit from Cell Signaling Technology (CST) 8818S, according to the manufacturer’s instructions, and western blot analysis.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot