rap1 assay  (Cell Signaling Technology Inc)

Journal: Biomedicines

Article Title: GDF6 Alleviates Pathological Cardiac Hypertrophy via AMPKα Signaling Pathway

doi: 10.3390/biomedicines13122935

Figure Lengend Snippet: GDF6 activates AMPKα through cyclic AMP/exchange protein directly activated by cAMP 1 (cAMP/Epac1) pathway. ( A ) The levels of cAMP and protein kinase A (PKA) activity in PE-stimulated NRVMs with or without GDF6 knockdown. ( B ) The levels of cAMP and PKA activity in PE-stimulated NRVMs with or without rhGDF6 treatment. ( C ) Quantification of cell area in rhGDF6-treated NRVMs with or without adenylyl cyclase (AC) inhibition. ( D ) Rap1-GTP and total Rap1 protein levels in PE-stimulated NRVMs treated with si Gdf6 or rhGDF6. ( E ) AMPKα protein levels in PE-stimulated NRVMs with or without Epac1 silence in the presence or absence of rhGDF6. ( F ) Quantification of cell area. ( G ) Epac1 mRNA levels in NRVMs with or without Epac1 silence. ( H ) Quantification of cell area. ( I ) Epac1 mRNA levels in murine hearts with or without Epac1 silence. ( J ) HW/TL in TAC mice with or without Epac1 silence in the presence or absence of AAV9- Gdf6 . ( K ) Quantification of CSA. ( L ) IVSd and IVSs. ( M ) FS, LVEDd, and LVEDs. n = 6 per group. * p < 0.05 versus matched AAV9- Ctrl -injected TAC mice receiving si Ctrl injection, # p < 0.05 versus matched AAV9- Gdf6 -injected TAC mice receiving si Ctrl injection. In ( A – D , G – I ), * p < 0.05 versus the matched groups. In ( E , F ), * p < 0.05 versus matched si Ctrl -transfected NRVMs with DMSO treatment under PE incubation, # p < 0.05 versus matched si Ctrl -transfected NRVMs with rhGDF6 treatment under PE incubation. One-way ANOVA followed by Tukey post hoc test was applied. In ( A , B , D , G – I ), Student’s two-tailed t -test was performed.

Article Snippet: Active Rap1 Detection Kit (#8818) was purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Knockdown, Inhibition, Injection, Transfection, Incubation, Two Tailed Test

Journal: World Journal of Gastrointestinal Oncology

Article Title: Downregulation of uncoupling protein 1 by hypermethylation in gastric cancer activates Rap1 signaling

doi: 10.4251/wjgo.v17.i9.108760

Figure Lengend Snippet: Uncoupling protein 1 regulates the Rap-1 signaling pathway in gastric cancer cells. A: Volcano plot for differential gene expression; B: The Kyoto Encyclopedia of Genes and Genomes enrichment of the upregulated genes in the low- Uncoupling protein 1 (UCP1)-expression group; C: Western blot for active and total Rap1 in gastric cancer cells with UCP1 knockdown (left) or overexpression (right). UCP1: Uncoupling protein 1; Ctrl: Control; OE: Overexpression.

Article Snippet: Active Rap1 (GTP-bound) was detected using an active Rap1 detection kit (Cell Signaling Technology, United States).

Techniques: Gene Expression, Expressing, Western Blot, Knockdown, Over Expression, Control

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