active rap1a detection kit  (Cell Signaling Technology Inc)

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 reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A) Rapgef4 (Epac2), G6pc , and Pck1 mRNA levels were analyzed from forskolin and dexamethasone (F + D)-treated primary mouse hepatocytes (HCs) that were transfected with scrambled RNA (scr) or siRNA against Epac2 (si-Epac2) (n = 4 biological replicates, mean ± SEM, *p < 0.05). (B) Same as in (A), except that glucose production was measured (n = 4 biological replicates, mean ± SEM, *p < 0.05). (C–E) Rap1a and β-actin levels (C), G6pc and Pck1 mRNA (D), and glucose production (E) from F + D-treated primary mouse hepatocytes that were transfected with scrambled RNA (scr) or siRNA against Rap1a (si-Rap1a) (n = 3–4 biological replicates, mean ± SEM, *p < 0.05). (F) G6pc and Pck1 mRNA levels were measured from glucagon-treated WT or Rap1a −/− mouse hepatocytes (n = 6 biological replicates, mean ± SEM, *p < 0.05). (G–L) Hepatic Rap1a and β-actin levels (G), body weight before and after AAV injection (H), 5-h fasting blood glucose (I), liver Pck1 mRNA (J), 5-h fasting plasma insulin (K), and glucose tolerance test (L) from DIO Rap1a fl/fl mice that were injected with adeno-associated viruses containing either hepatocyte-specific TBG-Cre recombinase (TBG-Cre) or the control vector (TBG-Gfp) (n = 5–8 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant). See also and .

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Transfection, Injection, Clinical Proteomics, Control, Plasmid Preparation

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A) Livers from db/db and control ( db /+) mice were assayed for GTP-bound (active) Rap1a and total Rap1a. Densitometric quantification of the immunoblot data is shown in the bar graph (n = 5 mice/group, mean ± SEM, *p < 0.05). (B) Livers from DIO mice and their low-fat-fed controls (lean) were assayed for GTP-bound (active) Rap1a, total Rap1a, and β-actin. Densitometric quantification of the immunoblot data is shown in the bar graph (n = 6 mice/group, mean ± SEM, *p < 0.05). (C–G) Body weight before and after AAV injection (C), overnight fasting blood glucose (D), glucose tolerance test (E), liver G6pc and Pck1 mRNA (F), and overnight fasting plasma insulin (G) from db/db mice that were injected with adeno-associated viruses (AAV) containing either hepatocyte-specific CA-Rap1a (constitutively active Rap1a) or the control vector (Gfp) (n = 4–7 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant). (H–L) Body weight before and after adenovirus injection (H), 5-h fasting blood glucose (I), glucose tolerance test (J), liver G6pc mRNA (K), and 5-h fasting plasma insulin (L) levels from DIO mice that were injected with adenovirus vectors containing CA-Rap1a or control LacZ (β-galactosidase) (n = 6 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant).

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Control, Western Blot, Injection, Clinical Proteomics, Plasmid Preparation

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A) Primary mouse hepatocytes (HCs) were treated with vehicle or 10 μM simvastatin (simva) for 20 h. Membrane proteins were assayed for Rap1a and Na, K-ATPase (loading control) (n = 2 biological replicates). (B) Same as in (A), except that 5 μM rosuvastatin (rosuva) was used, and membrane fractions and whole-cell lysates were assayed for Rap1a and loading controls (Na, K-ATPase and μ-actin, respectively) (n = 3 biological replicates). (C) Hepatocytes treated as in (B) were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 2 biological replicates). (D) Liver tissue samples from patients on statin therapy and their sex-, age-, and disease-matched controls without statin therapy were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 4 human liver samples/group). (E) Same as in (D), except that livers from another set of patients were assayed for GTP-bound (active) Rap1a and total Rap1a (n = 2 human liver samples/group).

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Membrane, Control

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A and B) Primary mouse hepatocytes (HCs) were treated with vehicle or 10 μM simvastatin (simva) for 24 h. The cells were then assayed for forskolin and dexamethasone (F + D)-induced G6pc and Pck1 mRNA (A) and glucose production (B) (n = 4 biological replicates, mean ± SEM, *p < 0.05). (C and D) F + D-induced G6PC (C) and PCK1 (D) mRNAs were measured from human primary hepatocytes treated with vehicle control or 5 μM rosuvastatin (rosuva) for 24 h (n = 4 biological replicates, mean ± SEM, *p < 0.05). (E and F) Primary mouse hepatocytes that were transfected with scrambled control (scr) or siRNA against Rap1a (si-Rap1a) were treated with vehicle or 5 μM rosuvastatin (rosuva) for 24 h. F + D-induced G6pc (E) and Pck1 mRNA levels (F) were assayed (n = 3 biological replicates, mean ± SEM, *p < 0.05, n.s., non-significant). (G–L) Body weight before and after the simvastatin treatment (G), 5-h fasting blood glucose (H), liver G6pc (I) and Pck1 (J) mRNA, glucose tolerance test (K), and 5-h fasting plasma insulin (L) levels from DIO mice that were fed with a high-fat diet containing 0.02% simvastatin (w/w) (simva) for 12 weeks (n = 6–7 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant). See also .

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Control, Transfection, Clinical Proteomics

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A and B) Primary mouse hepatocytes (HCs) were treated with vehicle, 10 μM simvastatin (simva), or simvastatin + geranylgeranyl pyrophosphate (GGPP) (10 μM) for 24 h. Membrane proteins were assayed for Rap1a and pan-cadherin (pan-CDH, loading control) (A), and forskolin and dexamethasone (F + D)-induced G6pc and Pck1 mRNA levels were measured (B) (n = 2–3 biological replicates, mean ± SEM, *p < 0.05). (C–G) DIO mice were fed with a high-fat diet containing 0.02% simvastatin (w/w) for 12 weeks. Mice were then administered with geranylgeraniol (GGOH, 100 mg/kg/day) or vehicle control by daily gavage for 3 weeks while still receiving the statin-containing diet. Overnight fasting blood glucose (C), glucose tolerance test (D), liver G6pc and Pck1 mRNA (E), body weight before and after simvastatin and GGOH treatment (F), and overnight fasting plasma insulin (G) were assayed (n = 5–7 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant). (H) Primary mouse hepatocytes that were transfected with si-Pggt1b (encoding GGT1) or scrambled control (scr) were assayed for Pggt1b , and F + D-induced G6pc and Pck1 mRNA levels (n = 3 biological replicates, mean ± SEM, *p < 0.05). (I–L) Liver Pggt1b mRNA (I), 5-h fasting blood glucose (J), body weight before and after AAV injection (K), and 5-h fasting plasma insulin (L) levels were assayed from DIO mice that were injected with AAV8 vectors containing shRNA against Pggt1b (sh-Pggt1b) or empty, control AAV8 (Con) (n = 7 mice/group, mean ± SEM, *p < 0.05, n.s., non-significant). See also – .

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Membrane, Control, Clinical Proteomics, Transfection, Injection, shRNA

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A and B) Igfbp1 mRNA levels were measured from the livers of DIO Rap1a fl/fl mice that were injected with TBG-Cre or TBG-Gfp (A), and vehicle- or 5 μM rosuvastatin (rosuva)-treated primary hepatocytes (HCs) that were incubated with forskolin and dexamethasone (F + D) (B) (n = 7–8 mice/group and n = 3 biological replicates, respectively, mean ± SEM, *p < 0.05). (C) Gck mRNA levels were measured from the livers of DIO Rap1a fl/fl mice that were injected with TBG-Cre or TBG-Gfp (n = 7–8 mice/group, mean ± SEM, *p < 0.05). (D) WT and Rap1a −/− primary hepatocytes were transfected with a luciferase fusion construct encoding nucleotides −1,227 to +57 of the G6pc promoter containing an intact FoxO binding site and FoxO1-Gfp or control plasmid. Relative luciferase activity and Gfp levels from WT and Rap1a −/− cells transfected with FoxO1-Gfp were measured (lower blots) (n = 3–4 biological replicates, mean ± SEM, *p < 0.05). (E) Luciferase reporter assay of the Igfbp1 promoter in WT and Rap1a −/− cells transfected with control or FoxO1-Gfp. Gfp levels from WT and Rap1a −/− cells transfected with FoxO1-Gfp were assayed in the lower blots (n = 3–4 biological replicates, mean ± SEM, *p < 0.05). (F) G6pc and Igfbp1 mRNA levels from F + D-treated WT and Foxo1 , 3 , 4 −/− cells that were transfected with scrambled RNA (scr) or siRNA against Rap1a (si-Rap1a) (n = 4 biological replicates, mean ± SEM, *p < 0.05). (G) Nuclear and whole-cell FoxO1 levels along with loading controls (lamin A/C and β-actin, respectively) in WT versus Rap1a −/− hepatocytes. Densitometric quantification of the nuclear FoxO1 immunoblot data is shown in the bar graph (n = 3 biological replicates). (H) Nuclear and whole-cell FoxO1 levels along with loading controls (nucleophosmin, Np, and β-actin, respectively) in vehicle or 5 μM rosuvastatin (rosuva)-treated cells (n = 3 biological replicates). (I) WT and Rap1a −/− hepatocytes were stimulated with vehicle control or insulin (100 nM) for 5 min, and p-Akt and total Akt levels were assayed (upper blots). Basal levels of phospho-Akt and total Akt in WT and Rap1a −/− cells are shown in the lower blots (n = 3 biological replicates). (J) Same as in (I) except that p-FoxO1, total FoxO1, and β-actin levels were measured (n = 3 biological replicates). (K–M) Vehicle or 5 μM rosuvastatin-treated cells were incubated with or without insulin (100 nM) for 5 min as indicated. p-Akt and total Akt (K), and p-FoxO1, total FoxO1, and β-actin levels (L–M) were assayed (n = 3 biological replicates). See also .

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Injection, Incubation, Transfection, Luciferase, Construct, Binding Assay, Control, Plasmid Preparation, Activity Assay, Reporter Assay, Western Blot

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: (A) Primary mouse hepatocytes (HCs) that were treated with scrambled control (scr), si-Rap1a, or cytochalasin D were stained with 488-phalloidin (green) to visualize F-actin. White arrows indicate fragmented F-actin filaments that form cytoskeletal clumps. Scale bar, 10 μm. (B) Hepatocytes were treated with vehicle, 0.1 μM, or 1 μM cytochalasin D (cyto. D) for 8 h, and forskolin and dexamethasone (F + D)-stimulated G6pc and Pck1 mRNA levels were measured (n = 4 biological replicates, mean ± SEM, *p < 0.05). (C) Same as in (B) except that glucose production was measured in cells treated with vehicle or 0.1 μM cytochalasin D (cyto. D) (n = 3 biological replicates, mean ± SEM, *p < 0.05). (D) Hepatocytes were treated with 0.1 μM jasplakinolide (jasp.) for 6 h, and F + D-stimulated G6pc and Pck1 mRNA levels were measured (n = 3 biological replicates, mean ± SEM, *p < 0.05). (E) Same as in (D), except that glucose production was measured (n = 4 biological replicates, mean ± SEM, *p < 0.05). (F) WT and Foxo1,3,4 −/− hepatocytes were treated with vehicle or 1 μM cytochalasin D for 8 h, and F + D-stimulated G6pc and Igfbp1 mRNA levels were measured (n = 4 biological replicates, mean ± SEM, *p < 0.05, n.s., non-significant). (G) Nuclear and whole-cell FoxO1 levels along with loading controls (nucleophosmin, Np, and β-actin, respectively) were assayed in hepatocytes that were treated with vehicle or 1 μM cytochalasin D for 8 h (n = 3 biological replicates). (H) Same as in (G), except that cells were stimulated with insulin, and p-Akt, total Akt, and β-actin levels were assayed (n = 3 biological replicates).

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Control, Staining

Journal: Cell reports

Article Title: Hepatocyte Rap1a contributes to obesity- and statin-associated hyperglycemia

doi: 10.1016/j.celrep.2022.111259

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rap1a activity was assayed using Active Rap1 Detection Kit (Cell Signaling Technology, cat # 8818).

Techniques: Virus, Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Reporter Assay, Negative Control, Luciferase, Software