Journal: Scientific reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells.

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Fig. 5. Rap1 overexpression reverses the expression of key proteins in CD147 knockdown mediated cell proliferation, apoptosis and EMT. (A, B) Western blot analysis of Rap1 and Rap1GAP protein levels in HCT116 and SW620 cells after CD147 knockdown (shCD147). β-actin served as a loading control. Data represent mean ± SD (n = 3; **P < 0.01, ***P < 0.001 vs. shNC, Student’s t-test). Original blots are presented in Supplementary Fig. 3. (C) qRT-PCR validation of Rap1 mRNA overexpression in CD147-knockdown cells. Expression normalized to β-actin (n = 3; ***P < 0.001 vs. HCT116 or SW620, Student’s t-test). (D, E) Western blot analysis of c-Myc, Bcl-2, Bax, N-cadherin, and E-cadherin protein levels in CD147-Knockdown cells with Rap1 overexpression (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. shNC, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. shCD147, one-way ANOVA). Original blots are presented in Supplementary Fig. 4.

Article Snippet: Supplier Antibody dilution used WB/IF Anti CD147 mouse monoclonal antibody 66443-1-AP ProteinTech Group, Inc. 1:5000 /1:200 Anti N-cadherin rabbit polyclonal antibody 22018-1-AP ProteinTech Group, Inc. 1:5000 Anti E-cadherin rabbit polyclonal antibody 20874-1-AP ProteinTech Group, Inc. 1:10000 Anti c-myc mouse monoclonal antibody 67,447-AP ProteinTech Group, Inc. 1:5000 Anti RAP1GAP rabbit polyclonal antibody 19174-1-AP ProteinTech Group, Inc. 1:2000 Anti RAP1 rabbit polyclonal antibody 14595-1-AP ProteinTech Group, Inc. 1:1000 Anti β-actin rabbit polyclonal antibody 20536-1-AP ProteinTech Group, Inc. 1:8000 HRP-conjugated Goat Anti-Mouse IgG(H + L) SA00001-1 ProteinTech Group, Inc. 1:10000 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) ab150116 Abcam 1:200 Table 1.

Techniques: Over Expression, Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Scientific reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells.

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Fig. 7. Rap1 restores CD147-mediated migration and invasion in colorectal cancer cells. (A, C) Scratch wound healing assay in HCT116 cells. Healing rates were quantified at 24 h and 48 h (n = 3; ***P < 0.001 vs. shNC, ##P < 0.01, ###P < 0.001 vs. shCD147, one-way ANOVA). (B, D) Transwell migration and Matrigel invasion assays. Migrated/invaded cells were counted and normalized to shCtrl (n = 3; ***P < 0.001 vs. shNC, ###P < 0.001 vs. shCD147, one-way ANOVA). SW620 cells showed consistent trends. (E) Mechanistic diagram of CD147 promoting tumor progression through Rap1/Rap1GAP signaling in colorectal cancer cells.

Article Snippet: Supplier Antibody dilution used WB/IF Anti CD147 mouse monoclonal antibody 66443-1-AP ProteinTech Group, Inc. 1:5000 /1:200 Anti N-cadherin rabbit polyclonal antibody 22018-1-AP ProteinTech Group, Inc. 1:5000 Anti E-cadherin rabbit polyclonal antibody 20874-1-AP ProteinTech Group, Inc. 1:10000 Anti c-myc mouse monoclonal antibody 67,447-AP ProteinTech Group, Inc. 1:5000 Anti RAP1GAP rabbit polyclonal antibody 19174-1-AP ProteinTech Group, Inc. 1:2000 Anti RAP1 rabbit polyclonal antibody 14595-1-AP ProteinTech Group, Inc. 1:1000 Anti β-actin rabbit polyclonal antibody 20536-1-AP ProteinTech Group, Inc. 1:8000 HRP-conjugated Goat Anti-Mouse IgG(H + L) SA00001-1 ProteinTech Group, Inc. 1:10000 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) ab150116 Abcam 1:200 Table 1.

Techniques: Migration, Wound Healing Assay

Journal: Molecular and cellular biochemistry

Article Title: Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

doi: 10.1007/s11010-019-03520-z

Figure Lengend Snippet: cAMP binding to its chemoattractant receptor cAMP receptor 1 (cAR1), coupled to the heterotrimeric G protein Gα2βγ, induces the activation of multiple signaling cascades, including the RasC, Rap1, and RasG-mediated pathways. The chemotactic effectors phosphatidylinositol 3-kinase (PI3K) and mechanistic Target of Rapamycin Complex 2 (mTORC2) play central roles in the cAMP chemoattractant signaling network. The different signaling cascades interact through crosstalks and many of the components are also linked through feedback loops. ACA, adenylyl cyclase A. CRAC, cytosolic regulator of adenylyl cyclase. ERK1, extracellular regulated kinase 1. ERK2, extracellular-regulated kinase 2. GbpC, cGMP binding protein C. GbpD, cGMP binding protein D. GCA, guanylyl cyclase A. GEFR, guanine nucleotide exchange factor R. GflB, guanine nucleotide exchange factor-like protein B. MyoII, myosin II. F-actin, filamentous actin. Phg2, phagocytosis protein 2. PKA, protein kinase A. PKB, protein kinase B. PKBR1, PKB-related kinase 1. Sca1C, scaffold 1 complex.

Article Snippet: The Rap1 (directed against amino acids 169–182 of Dictyostelium Rap1) was custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Binding Assay, Activation Assay

Journal: Molecular and cellular biochemistry

Article Title: Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

doi: 10.1007/s11010-019-03520-z

Figure Lengend Snippet: cAMP-induced activation of RasC (a), RasG (b), and Rap1 (c) in control and caffeine-treated cells. Active RasC, RasG, and Rap1, were pulled down with GST-Byr2(RBD), GST-Raf1(RBD), and GST-RalGDS(RBD), respectively, and revealed by immunoblotting. For RasC, cells expressing FLAG-RasC were used and RasC was revealed by immunoblotting using anti-FLAG antibody. Pan-Ras antibody was used to reveal RasG, and our custom Rap1 antibody (described previously [17]) was used to reveal Rap1. Immunoblots shown are representative of at least three independent experiments. Graphs show quantified data from three RasC, five RasG, and three Rap1 experiments, expressed as percentage of the maximal response in control cells, along with the means ± SD.

Article Snippet: The Rap1 (directed against amino acids 169–182 of Dictyostelium Rap1) was custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Activation Assay, Control, Western Blot, Expressing

Journal: Molecular and cellular biochemistry

Article Title: Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

doi: 10.1007/s11010-019-03520-z

Figure Lengend Snippet: Our findings suggest that caffeine inhibits mTORC2 and PI3K in Dictyostelium, leading to reduced activation of PKB, PKBR1, and cAMP production. We then propose that the caffeine inhibition of these two pathways lead to the observed reduced PKA activity and, consequently, the increased activity of Rap1 and RasG, which are negatively regulated by PKA [17]. The cAMP-stimulated production of cGMP is also considerably inhibited by caffeine, but through an unknown mechanism. In turn, the decrease in cGMP together with the increased Rap1 activity likely contributes to the observed decrease in MyoII assembly in caffeine-treated cells. Intriguingly, we obtained conflicting results concerning the effect of caffeine on F-actin polymerization depending on the assay used, and on ERK2 activity depending on the Dictyostelium strain used (depicted by the dual color). Finally, ERK1 activity is reduced and the cAMP-induced activation of the heterotrimeric G protein Gα2βγ is potentiated in caffeine treated cells, both through unknown mechanisms, whereas RasC activity is unaffected. A color scheme is used to depict the effects of caffeine on the chemotactic effectors, with dark red signifying the most inhibitory and dark blue the potentiation effects.

Article Snippet: The Rap1 (directed against amino acids 169–182 of Dictyostelium Rap1) was custom-made by ProSci Incorporated (Poway, CA, USA).

Techniques: Activation Assay, Inhibition, Activity Assay

Journal: Journal of translational medicine

Article Title: N-terminal domain of CTRP9 promotes cardiac fibroblast activation in myocardial infarction via Rap1/Mek/Erk pathway.

doi: 10.1186/s12967-025-06274-z

Figure Lengend Snippet: Fig. 5 nCTRP9 promotes cardiac fibrosis through upregulation of Rap1. A. Heatmap of the different genes after nCTRP9 treated immortalized cardiac fibroblasts (iCF) activated with Tgf-β. B KEGG pathway enrichment of differentially expressed genes. C. ESI-05, an inhibitor of Rap1, down-regulated the transcription of fibrosis-related genes in iCF. n = 4. D. nCTRP9 increased MEK 1/2 and ERK 1/2 phosphorylation in fibroblasts. n = 4. E. nCTRP9 increased MEK 1/2 and ERK 1/2 phosphorylation in mice heart post MI surgery. n = 4. F. Rap1 knockdown efficiency in iCF. n = 4. G. Down-regulation of Rap1 attenu ated the promoting effect of nCTRP9 on the transcription of fibrosis-related genes in iCF. n = 4. H. Rap1 knockdown attenuated α-smooth muscle actin (α-SMA) expression increased by nCTRP9 in iCF. n = 6. I. Rap1 knockdown attenuated the promoting effect of nCTRP9 on cell proliferation of iCF. n = 6. * p < 0.05, ** p < 0.01

Article Snippet: The following are the primary antibodies: CTRP9 (1:1000, customized by Genscript), nCTRP9 (1:1000, customized by Genscript), Rap1 (1:1000, 68125-1-Ig, Proteintech, Wuhan, China) GAPDH (1:5000, AC001, Abclonal, Wuhan, China), AMPK (1:1000, 2532, CST, Danvers, USA) and p-AMPKαThr172 (1:1000, 2532, CST, Danvers, USA), Phospho-p38 MAPKαThr 172 (1:1000, 4060, CST, Danvers, USA), Akt (1:1000, 9272, CST, Danvers, USA)and p-Akt Ser 473 (1:1000, 4060, CST, Danvers, USA), ERK1/2 (1:1000, 4696, CST, Danvers, USA), p-ERK1/2 Thr202/Tyr204 (1:1000, 9106, CST, Danvers, USA), MEK1/2 (1:1000, 11049-1-AP, Proteintech, Wuhan, China)and Phospho-MEK1/2(1:1000, 9154, CST, Danvers, USA).

Techniques: Phospho-proteomics, Knockdown, Expressing

Journal: Journal of translational medicine

Article Title: N-terminal domain of CTRP9 promotes cardiac fibroblast activation in myocardial infarction via Rap1/Mek/Erk pathway.

doi: 10.1186/s12967-025-06274-z

Figure Lengend Snippet: Fig. 6 RAP1 knockdown abolishes cardiac fibrosis induced by nCTRP9. (A) Fibroblast-specific knockdown of Rap1 mice was established by administrat ing adeno-associated virus (type 9) carrying the shRNA of Rap1 via tail vein injection. nCTRP9 was administered after MI surgery for 4 weeks. (B) Cardiac ejection fraction (EF) of mice post MI surgery. n = 6. (C) Infarct size of MI mice heart was detected after surgery for 4 weeks. n = 6. (D) Heart weight (HW) to tibial length (TL) ratio. n = 6. (E) Staining with wheat germ agglutinin was done to determine the mean cross-sectional area of cardiomyocytes. n = 6. F-G. Cardiac fibrosis in Sham and MI mice heart detected by Masson staining of border region and distant region. n = 6. H. Rap1 levels in MI mice heart. n = 4. I. Knockdown of Rap1 attenuated phosphorylation increase by nCTRP9 in MI mice heart. n = 4. * p < 0.05, ** p < 0.01

Article Snippet: The following are the primary antibodies: CTRP9 (1:1000, customized by Genscript), nCTRP9 (1:1000, customized by Genscript), Rap1 (1:1000, 68125-1-Ig, Proteintech, Wuhan, China) GAPDH (1:5000, AC001, Abclonal, Wuhan, China), AMPK (1:1000, 2532, CST, Danvers, USA) and p-AMPKαThr172 (1:1000, 2532, CST, Danvers, USA), Phospho-p38 MAPKαThr 172 (1:1000, 4060, CST, Danvers, USA), Akt (1:1000, 9272, CST, Danvers, USA)and p-Akt Ser 473 (1:1000, 4060, CST, Danvers, USA), ERK1/2 (1:1000, 4696, CST, Danvers, USA), p-ERK1/2 Thr202/Tyr204 (1:1000, 9106, CST, Danvers, USA), MEK1/2 (1:1000, 11049-1-AP, Proteintech, Wuhan, China)and Phospho-MEK1/2(1:1000, 9154, CST, Danvers, USA).

Techniques: Knockdown, Virus, shRNA, Injection, Staining, Phospho-proteomics

Journal: Journal of ethnopharmacology

Article Title: Anemarrhena asphodeloides Bunge and Phellodendri Chinensis Cortex inhibits the PTGS2/EP2/cAMP/Epac1 signaling pathway to reduce microglial M1 polarization, thereby blocking chronic stress-induced depression-like behavior.

doi: 10.1016/j.jep.2025.119792

Figure Lengend Snippet: Fig. 8. Neomangiferin alleviates the release of proinflammatory factors in BV2 cells by inhibiting PTGS2/EP2/cAMP/Epac1 signals. (A) representative immunofluorescence staining of EP2 and Epac1, and (B) fluorescence area (n = 3). (C–D) Protein expression changes of EP2 and Epac1 (n = 3). The contents of PGE2 and cAMP in BV2 cells were determined by E-F Elisa kit (n = 3). (G) Representative images of zebrafish embryos in Neomangiferin experiment (n = 10). Data are expressed as mean±SD. *** means P < 0.001,** means P < 0.01,* means P < 0.05, ns means P > 0.05.

Article Snippet: Seal with 5 % skim milk powder at room temperature for 2 h. Primary antibody β-actin (rabbit, 1:5000; bs-00R,Bioss, China), GAPDH(rabbit, 1:10,000; 10494- 1-AP,Proteintech, China), HSP90(rabbit, 1:5000; 13171-1-AP,Proteintech, China), Arg-1 (rabbit, 1:1000; 16001-AP, Proteintech, China), iNOS (rabbit, 1:1000; ab283655, Abcam, UK), PTGS2 (mouse, 1:400; 66351-1-Ig, Proteintech, China), EP2 (rabbit, 1:1000; HA721380, HUABIO, China), EPAC1 (rabbit, 1:1000; A02483-3, Boster, China), incubated overnight at 4 ◦C.

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Enzyme-linked Immunosorbent Assay