rap1 rabbit mab antibody  (ZenBio)

Journal: Antioxidants

Article Title: 3,6′-Disinapoyl Sucrose from Polygalae Radix Exerts Anti-Aging Effects via Modification of Telomeres, SIRT1/p53/p21 Pathway, Oxidative Stress and Autophagy

doi: 10.3390/antiox15030313

Figure Lengend Snippet: Effect of DISS on telomere. ( A ) The changes in telomerase content after treatment with 0, 1, 3, 10 μM DISS or 10 μM RES. ( B ) Relative telomere length of 3T3 cells after treatment with 0, 1, 3, 10 μM DISS or 10 μM AST. ( C , D ) Effects of 1, 3, 10 μM DISS or 10 μM RES on EST1 , EST2 genes at 24 and 48 h. ( E ) Effects of 0, 1, 3, 10 μM DISS or 10 μM AST on TRF2 protein expressions and digital results. ( F ) Effects of 0, 1, 3, 10 μM DISS or 10 μM AST on RAP1 protein expressions and the digital results. qPCR was performed for 40 cycles. For qPCR, each RNA sample was subjected to three technical replicates ( n = 3). Western blot analysis was performed with three biological replicates. ( n = 3). *, ** and *** represent significant differences at p < 0.05, p < 0.01, and p < 0.001 compared with the negative control group, respectively. # and ### represent significant differences at p < 0.05, p < 0.001 compared with the negative control group for 48 h, respectively.

Article Snippet: The first antibodies used in this experiment were as follows: SIRT1 antibody (Cell Signaling Technology, Shanghai, China, #9475, 1:1000), GFP antibody (Wuhan Sanying Biotechnology Co., Ltd., Wuhan, China, 50430-2-AP, 1:1000), p53 antibody (Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China, HA722074, 1:1000), p21 antibody (Hangzhou Huaan Biotechnology Co., Ltd., Hangzhou, China, HA722065, 1:1000), TRF2 antibody (Cell Signaling Technology, Shanghai, China, #13136, 1:1000), and RAP1 antibody (Cell Signaling Technology, Shanghai, China, #5433, 1:1000), and β -actin antibody (Hangzhou Daige Biotech Co., Ltd., Hangzhou, China, #db13986, 1:1000).

Techniques: Western Blot, Negative Control

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Berberine Overcomes Gemcitabine-Associated Chemoresistance through Regulation of Rap1/PI3K-Akt Signaling in Pancreatic Ductal Adenocarcinoma.

doi: 10.3390/ph15101199

Figure Lengend Snippet: Figure 4. Rap1/PI3K-Akt signaling pathway correlates with Gemcitabine-resistance in PDAC cells. (A) Schematic of the differentially expressed gene discovery in Gem-R PDAC cells using GSE148200 and GSE140077. (B) Venn-diagram of up- and down-regulated expression (log2FC > ±1.0 and p < 0.01) of the genes in Gem-R PDAC cells. (C,D) Scatter plot of KEGG pathway enrichment analysis of up-

Article Snippet: Primary antibodies against Rap1 (1:1000, #2399; Cell Signaling Technology [CST], Danvers, MA, USA), PI3K (1:500, #4249; CST), Akt (1:1000, #4691; CST), and phospho-Akt (Ser473) (1:1000, #4060; CST) were used.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium

doi: 10.3389/fcell.2021.687598

Figure Lengend Snippet: Endothelial dysfunction in Rap1i ΔEC mice is attenuated by L-arginine (L-Arg) supplementation. (A,B) Corrected vasorelaxation. Concentration dependent vasorelaxation of U466169-preconstricted aortae from control (WT) and Rap1 i Δ EC mice treated with or without L-arginine in response to ACh (A) , sodium nitroprusside (SNP) (B) . SNP response was measured in presence of L-NAME, an NO blocker. Values are means ± SEM ( n = 6). (C) L-arginine treatment normalizes blood pressure in Rap1 iΔEC mice. Systolic blood pressure was measured in control (WT) and Rap1 iΔEC mice with or without L-arginine supplementation in drinking water. Values are means + SEM ( n = 5–9).* indicates p < 0.05 vs. control without L-arginine; ** indicates p < 0.01 vs. Rap1 iΔEC without L-arginine. One-way ANOVA Tukey’s multiple comparison test was applied to compare statistical significance.

Article Snippet: Rap1 rabbit monoclonal antibody (clone 26B4, Cell Signaling Technologies #2399) was used for GTP-Rap1 pull-down assay.

Techniques: Concentration Assay, Control, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium

doi: 10.3389/fcell.2021.687598

Figure Lengend Snippet: (A,B) RaplB deletion inhibits agonist-induced NO release. NO release measured in HPAECs preincubated with 2 ml DAF-DA (5 μM) + L-arginine 10 μM with or without ACh (A) and bradykinin (BK) (B) for 30 min at 37°C. Mean values of concentrations calculated using authentic standards are shown ( n = 3). ND: not dected. * indicates p < 0.05 vs. siControl unstimulated. One-way ANOVA Tukey’s multiple comparison test was applied to compare statistical significance. (C,D) Rap1 is activated by GPCR agonist that induced nitric oxide (NO). Bradykinin (BK)-induced, GTP-bound Rap1 using Rap1-GTP pulldown assay. Representative immunoblot (C) and quantification (D) of Rap1 bound to GST-RalGDS beads following 1 and 5 min treatment with BK. Values are means ± SEM ( n = 3). * indicates p < 0.05 vs. 0’ BK. Unpaired t -test was applied to compare statistical significance. (E,F) Rap1B deletion attenuates agonist-induced Ser-1177 eNOS phosphorylation. Representative immunoblot (E) and quantification (F) of fold increase of phosphor-eNOS (Ser1177) vs.0 ATP in siControl and siRap1B HCAECs ( n = 4). * indicates p < 0.05 vs. siControl 2’ ATP. One-way ANOVA Tukey’s multiple comparison test was applied to compare statistical significance.

Article Snippet: Rap1 rabbit monoclonal antibody (clone 26B4, Cell Signaling Technologies #2399) was used for GTP-Rap1 pull-down assay.

Techniques: Comparison, Western Blot, Phospho-proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium

doi: 10.3389/fcell.2021.687598

Figure Lengend Snippet: Model: how Rap1A, Rap1B contribute to NO release, role in agonist-induced eNOS activation. Two Rap1 isoforms regulate eNOS activity via distinct signaling pathways; Rap1B promotes eNOS, activating Ser1177 phosphorylation, while Rap1A suppresses Thr495 eNOS phosphorylation, preventing eNOS inhibition. Candidate kinases regulated by Rap1 isoforms, PKC/AMPK/CaMKII, are indicated in blue font.

Article Snippet: Rap1 rabbit monoclonal antibody (clone 26B4, Cell Signaling Technologies #2399) was used for GTP-Rap1 pull-down assay.

Techniques: Activation Assay, Activity Assay, Protein-Protein interactions, Phospho-proteomics, Inhibition

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: Cytoplasmic RAP1 is an indicator of high-grade NSCLC. ( a and b ) Expression of cytoplasmic and nuclear RAP1 in lung adenocarcinoma ( a ) and squamous cell carcinoma ( b ) tissues, as well as peri-tumoral normal tissues (PTNT). ( c and d ) Expression of cytoplasmic RAP1 in Grade I–III adenocarcinoma ( c ) and squamous cell carcinoma ( d ) tissues. ( e ) Kaplan–Meier analysis and Log-rank (Mantel–Cox) test of the survival between RAP1-high ( n =33, expression >0.4) and RAP1-low ( n =59, expression <0.4) adenocarcinoma patients. ( f ) Representative images of RAP1 expression in adenocarcinoma, squamous cell carcinoma and PTNT, arrowhead: cytoplasmic RAP1; scale bar: 10 μ m. * P <0.05, ** P <0.01, *** P <0.001, Student’s t -test

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: Proliferation of NSCLC cells is inhibited after Rap1 deletion. ( a ) RAP1 mRNA expression in 16HBE and HSAEC1-KT (HSA for abbreviation in following) lung epithelial cells and A549, PC9 and HCC827 NSCLC cells measured by qRT-PCR. ( b ) Western blotting analysis for RAP1 in cytoplasmic and nuclear fractions of lung epithelial and NSCLC cells, representatives of three independent experiments. ( c ) Immunofluorescence (IF) detection of RAP1 (red) in 16HBE and NSCLC cells. Arrowhead: cytoplasmic RAP1; scale bar: 5 μ m. ( d ) RAP1 mRNA expression in A549, PC9 and HCC827 cells after transducing lentiviral vector coding two shRNAs against RAP1 (shRAP1) or scramble shRNA. ( e ) Proliferation of NSCLC cells after transduction with shRAP1 or scramble shRNA. Cells were plated in 96-well plates, cultured for 96 h and viability determined through cell counting kit-8 (CCK-8) assay. ( f ) Colony-formation assays for A549 cells after transduction with shRAP1 or scramble shRNA, detected by Giemsa staining. Statistics were generated from three independent experiments. ** P <0.01 *** P <0.001, Student’s t -test (two groups) or one-way analysis of variance with Bonferroni’s correction (three or more groups); error bar: ±S.D.

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Plasmid Preparation, shRNA, Transduction, Cell Culture, Cell Counting, CCK-8 Assay, Staining, Generated

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: RAP1 acts through NF- κ B signaling to support NSCLC cell growth. ( a and b ) Western blotting analysis ( a ) and IF assay ( b ) of NF- κ B signaling factors: p65, phosphorylated-p65 (pp65), I κ B α , and phosphorylated-I κ B α (p-I κ B α ) in the cytoplasmic and nuclear fractions of A549 cells transduced with shRAP1 or scramble shRNA. Scale bar: 5 μ m. ( c ) Expression of genes positively regulated by NF- κ B signaling in A549 cells transduced with shRAP1 or scramble shRNA. Statistics were generated from three independent experiments. ** P <0.01, *** P <0.001, Student’s t -test; error bar: ±S.D.

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Western Blot, Transduction, shRNA, Expressing, Generated

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: RAP1 desensitizes NSCLC cells to CP-induced cell death. ( a ) shRAP1- or scramble shRNA-transduced A549 cells were treated with different concentrations of CP for 48 h, and cell viability was determined with CCK-8 assay and normalized to the same type of cells cultured without CP. ( b ) Proliferation of A549 cells after transduction with control (CTRL) or RAP1-overexpressing (RAP1) lentiviral vectors. ( c ) Cell viability of A549 cells transduced with RAP1 or CTRL viruses and treated with different concentrations of CP for 48 h, after normalization to the same type of cells cultured in CP-free media. ( d ) Competition assay of the untransduced (GFP − ) cells co-cultured with the cells transduced with the indicated different RAP1 modification lentiviruses (GFP + ). Cells were treated with 2 μ M of CP and harvested at 24 and 72 h. Percentage of GFP + cells were analyzed for the viable population determined by 4,6-diamidino-2-phenylindole staining. ( e and f ) Untransduced (UnTxD) A549 cells and A549 cells with RAP1 overexpression (RAP1) or RAP1 knockdown (shRAP1) were cultured in media containing 1 μ M CP (+) or CP-free media (−) for 24 h and analyzed for the apoptotic status with western blotting analysis for cleaved caspase-3 (C-Cas3) and BCL-2 ( e ) or with flow cytometric analysis of Annexin-V ( f ), representatives of three independent experiments. Statistics were generated from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, Student’s t -test; error bar: ±S.D.

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: shRNA, CCK-8 Assay, Cell Culture, Transduction, Control, Competitive Binding Assay, Modification, Staining, Over Expression, Knockdown, Western Blot, Generated

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: CP resistance is associated with RAP1-dependent NF- κ B signaling induction. ( a ) Schema of stepwise CP treatment on A549 cells. ( b and c ) RAP1 expression and NF- κ B activation during CP treatment. A549 cells treated with CP as depicted in panel ( a ) were harvested at the indicated time points; the protein expression of RAP1, p65, pp65 and p-I κ B α in the cytoplasmic and nuclear fractions was measured with western blotting analyses ( b ), representatives of three independent experiments; and the mRNA expression of NF- κ B signaling downstream factors was analyzed with qRT-PCR ( c ). ( d ) A549 cells with overexpressed RAP1 were treated with CP as depicted in panel a . RAP1, pp65 and p-I κ B α were detected in the cytoplasmic (Cyto) and nuclear (Nu) fractions of cell lysate at the indicated time points, representatives of three independent experiments. ( e ) Cell viability of A549 cells transduced with shRAP1 or scramble shRNA at different time points during the sequential CP treatment as depicted in panel a , normalized to the same type of cells cultured in CP-free media in respective time point. ( f ) Nuclear (upper panel) and cytoplasmic (lower panel) fractions were isolated from A549 cells transduced with shRAP1 or scramble shRNA and treated with 0.5 μ M of CP for 24 h and assayed for p65, pp65 and p-I κ B α protein levels, representatives of three independent experiments. ( g ) mRNA expression of NF- κ B signaling downstream factors in shRAP1-transduced A549 cells after 24 h culture in the presence or absence of 0.5 μ M of CP. Statistics were generated from three independent experiments. ** P <0.01, *** P <0.001, Student’s t -test; error bar: ±S.D.

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Expressing, Activation Assay, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Cell Culture, Isolation, Generated

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: RAP1 deletion is lethal to CP-resistant NSCLC cells. ( a and b ) Protein expression of RAP1, pp65, p-I κ B α ( a ) and mRNA expression of NF- κ B signaling downstream factors ( b ) in control (CTRL), RAP1-overexpressed (RAP1) and CP-resistant (A549R) cells, representatives of three independent experiments. ( c ) Cell proliferation of A549R cells transduced with scramble shRNA or shRAP1 assayed with CCK-8 staining at the time point of transduction. Cells were precultured in CP-free media for 24 h before transduction. ( d ) RAP1-deleted A549 cells and scramble shRNA-transduced A549R cells were cultured in media containing 1 μ M CP (+) or CP-free media (−) for 24 h before being lysed to harvest protein. Lentiviral shRAP1 was introduced to A549R cells precultured in CP-free media for 24 h and protein harvested 12 h after transduction. Cleaved caspase-3 and BCL-2 were detected by western blotting. ( e ) IF assay of cleaved caspase-3 (red) in untreated A549 and A549R cells transduced with scramble shRNA or shRAP1. ( f ) Flow cytometric analysis for Annexin-V staining in A549 and A549R cells after RAP1 deletion. A549 cells were maintained in CP-free media and A549R cells were preconditioned in CP-free media for 24 h before analysis. Statistics were generated from three independent experiments. * P <0.05, ** P <0.01, *** P <0.001, Student’s t -test; error bar: ±S.D.

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Expressing, Control, Transduction, shRNA, CCK-8 Assay, Staining, Cell Culture, Western Blot, Generated

Journal: Cell Death & Disease

Article Title: Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer

doi: 10.1038/cddis.2017.210

Figure Lengend Snippet: Schematic depiction of the proposed model. CP generates DNA damage, which eventually leads to cell apoptosis. Meanwhile, RAP1 is upregulated after CP treatment, possibly through a direct or indirect induction by DNA damage response. The cytoplasmic fraction of RAP1 thus acts to facilitate the IKK-mediated activation of NF- κ B signaling, which subsequently induces transcription of downstream factors, including the apoptosis inhibitor BCL-2. Therefore, RAP1 functions through activating NF- κ B signaling to mediate resistance to CP

Article Snippet: Paraffin-embedded sections were immunolabeled with anti-Rap1 antibody (Cell Signaling Technology, Danvers, MA, USA #5433).

Techniques: Activation Assay