Journal: Oncogene

Article Title: Inhibition of farnesyltransferase increases TGFbeta type II receptor expression and enhances the responsiveness of human cancer cells to TGFbeta.

doi: 10.1038/sj.onc.1203920

Figure Lengend Snippet: Figure 3 FTI-277 enhances the ability of TGFb1 to induce transcription. (A) Panc-1 cells were cotransfected with 2 mg of p3TP-Lux reporter construct and 0.5 mg of pCMV-bgal. Fifteen hours post-transfection, cells were incubated with vehicle (DMSO), FTI-277 (15 mM), and/or TGFb1 (200 pM) for 24 h. The fold induction was calculated by dividing the luciferase activity values of FTI-277- or TGFb1-treated samples by that of vehicle-treated control. Data are means+s.e. for six experiments. (B) FTI-277 inhibits Lamin B farnesylation but not Rap1A geranylgeranylation. Cell lysates from experiment (A) above were analysed by SDS ± PAGE followed by immunoblotting with an anti-Lamin B or anti-Rap1A antibody. Lamin B is a substrate for farnesyltransferase and thus, its farnesylation was inhibited by FTI-277 (lanes 2 and 4). However, FTI-277 had no eect on Rap1A (lanes 2 and 4), which is a substrate for geranylgeranyl- transferase I. U for unprenylated and P for prenylated form. (C) Nuclear extracts (NE) from FTI-277- and TGFb1-treated samples were used in EMSA to determine the status of nuclear complexes that bound TRE sequence, similar to that present in p3TP-Lux reporter. The binding reactions contained NE from vehicle- treated samples and either 1006 of wild type competitor, TRE (lane 1) or 1006 of an unrelated oligonucleotide (lane 2). In addition to NE from speci®c samples, as indicated on top of the ®gure, the binding reactions contained either a normal rabbit antibody (lanes 3 ± 6) or an anti-AP1 antibody (lanes 7 ± 10). Note, cotreatment of cells with both FTI-277 and TGFb led to a higher level of TRE-bound complexes (lane 6) that were supershifted with an anti-AP1 antibody (lane 10). Data is representative of four independent experiments

Article Snippet: Brie ̄y, aliquots of cell lysate were analysed by SDS±PAGE followed by immunoblotting with anti-Lamin B antibody or, as control, anti-Rap1A antibody (sc-6216, sc-1482, respectively, SantaCruz Biotechnology).

Techniques: Construct, Transfection, Incubation, Luciferase, Activity Assay, Control, SDS Page, Western Blot, Sequencing, Binding Assay

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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: Anti-Rap1a antibody (cat # AF3767) was from R&D Systems.

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

Journal: Platelets

Article Title: Protein expression in platelets from six species that differ in their open canalicular system

doi: 10.3109/09537101003611385

Figure Lengend Snippet: The antibodies used and the sequence identities of the proteins examined.

Article Snippet: Antibodies and reagents Antibodies used in this study (see ) are as follows: an anti-Arf6 rabbit polyclonal antibody produced, under contract, by Bethyl Laboratory (Montgomery, TX, USA) and described in [ 17 ], an anti-RabGDI α rabbit polyclonal antibody generated and characterized in [ 23 ]; anti-Src pp60 , anti-IQGAP1, anti-Cdc42, anti-RalA, anti-Rap1A and anti-GIT1 mouse monoclonal antibodies (BD Biosciences, San Jose, CA, USA); anti-IQGAP2 mouse monoclonal antibody (GeneTex, San Antonio, TX), USA; anti-Rac1, anti-K/H/N-Ras and anti-calmodulin (CaM) mouse monoclonal antibodies (Upstate Biotechnology, Lake Placid, NY, USA); anti- β 3 integrin polyclonal antibody (Cell Signaling, Danvers, MA, USA); anti- α -actinin mouse monoclonal antibody and anti-Arf1/3 sheep polyclonal antibody (Sigma, St Louis, MO, USA); anti-focal adhesion kinase (FAK) rabbit polyclonal antibody (EMD Chemicals Inc., Gibbstown, NJ, USA); and anti-RhoA mouse monoclonal antibody (Cytoskeleton, Denver, CO, USA).

Techniques: Sequencing