rap1  (Proteintech)

Journal: Nature Communications

Article Title: Remodeling of the immune microenvironment is linked to adverse outcome in pediatric T cell acute lymphoblastic leukemia

doi: 10.1038/s41467-025-65134-y

Figure Lengend Snippet: a Bar graph of significant gene ontologies based on the top 100 differentially expressed genes in malignant cells of group 2 T-ALL patients compared to group 1 T-ALL patients (Supplementary Data ) as analyzed by DAVID Gene ontology. P -value is derived from a Modified Fisher’s Exact Test. Immunoreg: Immunoregulatory interactions between a lymphoid and non-lymphoid cell. b Ligand-receptor interactions (NATMI ) between NC monocytes and malignant cells from T-ALL group 2 patients. c , d Violin plots showing surface protein expression of CD54 in NC monocytes from healthy donors ( n = 102 cells (BM), n = 168 cells (PB)), T-ALL group 1 ( n = 881 cells (PB), n = 1531 cells (BM)) and T-ALL group 2 ( n = 1603 cells (PB), n = 1226 cells (BM)) at diagnosis ( c ), and CD11a and CD18 in malignant cells from T-ALL group 1 ( n = 4 (BM), n = 6 (PB)) and T-ALL group 2 ( n = 1 (BM), n = 4 (PB)) patients (downsampled to 300 cells/sample) at diagnosis ( d ). A one-way Anova test followed by Tukey’s HSD post-hoc test ( c ) or an unpaired t-test ( d ) was used for statistical analysis. e Viability as assessed by CellTiter-Glo assay upon GGTI-298 treatment in T-ALL cell lines (Jurkat, HPB-ALL and CCRF-CEM) for three days and two primary T-ALL samples from group 2 (P8 and P14) for five days. Viability is normalized to vehicle control DMSO. Each dot represents a data point from an independent replicate. Bars and error bars represent the mean and SEM of triplicates. f Heatmap of normalized cytochrome c - cells upon 12-hour treatment with GGTI-298 or vehicle DMSO in T-ALL cell lines Jurkat, HPB-ALL and CCRF-CEM. g Most synergistic area score (MSA) (Bliss, SynergyFinder ) of combination treatment with Rap1 inhibitor GGTI-298 and indicated drugs in T-ALL cell lines (Jurkat, HPB-ALL and CCRF-CEM) and two primary T-ALL patients samples from group 2. MSA > 10 is considered strongly synergistic (dashed line). Each dot represents the MSA and error bars indicate 95% confidence interval. h Dose-response curves (CellTiter-Glo assay) of T-ALL cell lines (Jurkat and HPB-ALL) and primary T-ALL P14 treated with various concentrations of GGTI-298 and navitoclax (top) or venetoclax (bottom) for three or five days, respectively. Viability of GGTI-298 without addition of BCL2 inhibitors were normalized and set to 100%. Most synergistic area score (MSA) (Bliss, SynergyFinder ) is shown (see ( g )). Each dot represents the mean and error bars represent SEM of three independent replicates.

Article Snippet: Cells were treated for 3 days (T-ALL cell lines) or 5 days (primary T-ALL cells) at indicated concentrations of Rap1 inhibitor GGTI298 (MedChemExpress, HY-15871), navitoclax (MedChemExpress, HY-10087) and venetoclax (MedChemExpress, HY-15531).

Techniques: Derivative Assay, Modification, Expressing, Biomarker Discovery, Glo Assay, Control

Journal: Nature Communications

Article Title: Remodeling of the immune microenvironment is linked to adverse outcome in pediatric T cell acute lymphoblastic leukemia

doi: 10.1038/s41467-025-65134-y

Figure Lengend Snippet: a Heatmap of scaled normalized RNA expression of top 15 genes differentially expressed in leukemia cells of group 1 T-ALL patients and group 2 T-ALL patients. A score for each group was computed based on the sum of expression of these genes. b Violin plots showing surface protein expression of genes present in the T-ALL transcriptomic scores shown in ( a ) for malignant cells from T-ALL group 1 ( n = 4 (BM), n = 6 (PB)) and T-ALL group 2 ( n = 1 (BM), n = 4 (PB)) patients (downsampled to 300 cells/sample) at diagnosis. An unpaired t-test was used for statistical analysis; n.s.: not significant. c Group 2 scores for primary T-ALL patient samples of an independent dataset ( GSE181157 ) split by percentage of CD4 - CD8 - CD7 - T cells analyzed by flow cytometry ( < 3% n = 11, >3% n = 3, see Supplementary Fig. ). A Wilcoxon rank-sum test was used for statistical analysis. d Visualization of T-ALL oncogenic groups ordered by group 2 scores for patient samples from the TARGET cohort ( n = 265). A chi-square test comparing lower tertile and upper tertile was used for statistical analysis. e Kaplan-Meier curve of event-free survival (EFS, n = 580) and overall survival (OS, n = 597) of MRD low T-ALL patients from the Gabriella Miller Kids First Pediatric Research Program dbGaP phs002276.v2.p1 grouped based on score for group 2. A log-rank test was used for statistical analysis to compare high versus low score groups, also see Supplementary Fig. . f Illustration of the remodeled immune system and leukemic Rap1 signaling in group 2 T-ALL patients.

Article Snippet: Cells were treated for 3 days (T-ALL cell lines) or 5 days (primary T-ALL cells) at indicated concentrations of Rap1 inhibitor GGTI298 (MedChemExpress, HY-15871), navitoclax (MedChemExpress, HY-10087) and venetoclax (MedChemExpress, HY-15531).

Techniques: RNA Expression, Expressing, Biomarker Discovery, Flow Cytometry

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Fu-Fang-Qi-Di-Hua-Yu-Tang Improves Diabetic Macrovascular Disease via PI3K/AKT Pathway Regulation

doi: 10.2147/DMSO.S515521

Figure Lengend Snippet: Comparison of mRNA expression via the RAP1/PI3K/AKT pathway among different groups. ( a ) Relative expression of RAP 1; ( b ) Relative expression of PI3K; ( c ) Relative expression of AKT; ( d ) Relative expression of mTOR. Compared with the control (** P< 0.01), model ( # P< 0.05, ## P< 0.01), L-FFQD ( ◆ P< 0.05, ◆◆ P< 0.01), M-FFQD ( Δ P< 0.05, ΔΔ P< 0.01), and H-FFQD ( ★ P< 0.05, ★★ P< 0.01) groups, significant differences were observed.

Article Snippet: The membrane was then incubated overnight at 4°C with primary antibodies: LC3-II/I (18725-1-AP, Proteintech, USA), NF-κB p65 (10,745-1-AP, Proteintech, USA) (1:500), RAP1 (10,840-1-AP, Proteintech, USA), PI3K (20584-1-AP, Proteintech, USA), P-PI3K (bs-6417R, Bioss, China), mTOR (#2983, CST, USA) (1:1000), P-mTOR (67778-1-Ig, Proteintech, USA) (1:2000), RAGE (16346-1-AP, Proteintech, USA), P-AKT (66444-1-Ig, Proteintech, USA) (1:3000), P62 (18,420-1-AP, Proteintech, USA), AKT (60203-2-Ig, Proteintech, USA), FOX01 (18,592-1-AP, Proteintech, USA), β-actin (66009-1-Ig, Proteintech, USA), and GAPDH (10494-1-AP, Proteintech, USA) (1:5000) antibodies.

Techniques: Comparison, Expressing, Control

Journal: Diabetes, Metabolic Syndrome and Obesity

Article Title: Fu-Fang-Qi-Di-Hua-Yu-Tang Improves Diabetic Macrovascular Disease via PI3K/AKT Pathway Regulation

doi: 10.2147/DMSO.S515521

Figure Lengend Snippet: Comparison of protein expression via the RAP1/PI3K/AKT pathway among different groups. ( a ) Electrophoresis of protein expression (A. Control group; B. Model group; C. L-FFQD group; D. M-FFQD group; E. H-FFQD group; F. ATOR-MAT group); ( b ) Relative value of RAP1; ( c ) Relative value of P-PI3K/PI3K; ( d ) Relative value of P-AKT/AKT; ( e ) Relative value of P-mTOR/mTOR; ( f ) Relative value of FOX01. Compared with the control (** P< 0.01), model ( # P< 0.05, ## P< 0.01), L-FFQD ( ◆ P< 0.05, ◆◆ P< 0.01), M-FFQD ( ΔΔ P< 0.01), and H-FFQD ( ★★ P< 0.01) groups, significant differences were observed.

Article Snippet: The membrane was then incubated overnight at 4°C with primary antibodies: LC3-II/I (18725-1-AP, Proteintech, USA), NF-κB p65 (10,745-1-AP, Proteintech, USA) (1:500), RAP1 (10,840-1-AP, Proteintech, USA), PI3K (20584-1-AP, Proteintech, USA), P-PI3K (bs-6417R, Bioss, China), mTOR (#2983, CST, USA) (1:1000), P-mTOR (67778-1-Ig, Proteintech, USA) (1:2000), RAGE (16346-1-AP, Proteintech, USA), P-AKT (66444-1-Ig, Proteintech, USA) (1:3000), P62 (18,420-1-AP, Proteintech, USA), AKT (60203-2-Ig, Proteintech, USA), FOX01 (18,592-1-AP, Proteintech, USA), β-actin (66009-1-Ig, Proteintech, USA), and GAPDH (10494-1-AP, Proteintech, USA) (1:5000) antibodies.

Techniques: Comparison, Expressing, Electrophoresis, Control

Journal: PLOS One

Article Title: Proteomic profiling of small extracellular vesicles from bovine nucleus pulposus cells

doi: 10.1371/journal.pone.0324179

Figure Lengend Snippet: A) STRING network of 141 more abundant NP small EV proteins; B) Cluster 1: Complement component C1q complex (red), complement and coagulation cascades (blue), complement activation classical pathway (green); C) Cluster 2: Proteasome (red); D) Cluster 3: Axon (blue), axon guidance receptor activity (red), Ephrin signaling (green); E) Cluster 4: Ras signaling (red), axon guidance (green) and MAPK signaling pathway (blue); F) Cluster 5: Hemostasis (green), regulation of MAPK cascade (blue), complement and coagulation cascades (red); G) Cluster 6: ECM (pink), integrin binding (light green), collagen binding (sky blue), ECM receptor interaction (red), PI3K/AKT signaling pathway (blue), ECM organization (yellow), signaling by RTKs (dark green). STRING: Search tool for the retrieval of interacting genes/proteins; NP: Nucleus pulposus.

Article Snippet: Environmental Information Processing , Regulation of actin cytoskeleton Focal adhesion Rap1 signaling pathway ECM-receptor interaction PI3K-Akt signaling pathway Chemokine signaling pathway Apelin signaling pathway Sphingolipid signaling pathway Ras signaling pathway cAMP signaling pathway MAPK signaling pathway , bta04810 bta04510 bta04015 bta04512 bta04151 bta04062 bta04371 bta04071 bta04014 bta04024 bta04010 , ACTN1, C9, COL6A1, COL6A3, FLNA, FN1, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GSN, HSPA8, HSPB1, HSPG2, ITGA3, ITGAV, ITGB1, LAMC1, MSN, MYH9, PFN1, RAB5B, RAB5C, RAC1, RHOA, RRAS, RRAS2, RAP1B, TLN1, YWHAQ, YWHAE, YWHAG, YWHAZ.

Techniques: Coagulation, Activation Assay, Activity Assay, Binding Assay

Journal: PLOS One

Article Title: Proteomic profiling of small extracellular vesicles from bovine nucleus pulposus cells

doi: 10.1371/journal.pone.0324179

Figure Lengend Snippet: A) STRING network of 484 NP small EV proteins; B) Cluster 1: Extracellular vesicle biogenesis (red), multivesicular body sorting pathway (blue), ESCRT I complex (pink), ESCRT (green), Flotillin complex (yellow); C) Cluster 2: Detection of oxidative stress (black), pentose phosphate pathway (red), glycolysis/gluconeogenesis (blue), biosynthesis of amino acids (green), carbon metabolism (pink), pyruvate metabolism (dark green), TCA cycle (yellow), glutathione metabolism (sky blue), HIF1 signaling pathway (purple), amino sugar and nucleotide sugar metabolism (ochre yellow), activation of BAD and translocation to mitochondria (brown), regulation of localization of FOXO transcription factors (grey); D) Cluster 3: Regulation of Schwann cell migration (blue), G-protein coupled receptor signaling pathway (green), axon guidance (red); E) Cluster 4: Rac protein signal transduction (red), RAS protein signal transduction (blue), small GTPase mediated signal transduction (green), RAP1 signaling pathway (yellow), EPHB-mediated forward signaling (pink), VEGFA/VEGFR2 pathway (dark green), signaling by Rho GTPases (sky blue); F); Cluster 5: MAP2K and MAPK activation (red), RAS signaling pathway (green), MAPK signaling pathway (pink), PI3K/AKT signaling pathway (yellow); G) Cluster 6 version 1: Cell adhesion mediated by integrin (red), mesodermal cell differentiation (blue), angiogenesis (green), regulation of small GTPase mediated signal transduction (yellow), negative regulation of apoptotic process (pink); H) Cluster 6 version 2: ECM receptor interaction (red), focal adhesion (green), PI3K/AKT signaling pathway (blue), axon guidance (yellow), TGFβ signaling pathway (pink); I) Cluster 7: Positive regulation of lamellipodium assembly (blue), actin cytoskeleton organization (red), EPHB mediated forward signaling (green); J) Cluster 8: ECM assembly (pink), cartilage development (green), angiogenesis (blue), blood vessel development (red), cell adhesion (yellow), PI3K/AKT signaling pathway (dark green). ECM: Extracellular matrix; ESCRT I: Endosomal sorting complex required for transport I; STRING: Search tool for the retrieval of interacting genes/proteins; NP: Nucleus pulposus.

Article Snippet: Environmental Information Processing , Regulation of actin cytoskeleton Focal adhesion Rap1 signaling pathway ECM-receptor interaction PI3K-Akt signaling pathway Chemokine signaling pathway Apelin signaling pathway Sphingolipid signaling pathway Ras signaling pathway cAMP signaling pathway MAPK signaling pathway , bta04810 bta04510 bta04015 bta04512 bta04151 bta04062 bta04371 bta04071 bta04014 bta04024 bta04010 , ACTN1, C9, COL6A1, COL6A3, FLNA, FN1, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GSN, HSPA8, HSPB1, HSPG2, ITGA3, ITGAV, ITGB1, LAMC1, MSN, MYH9, PFN1, RAB5B, RAB5C, RAC1, RHOA, RRAS, RRAS2, RAP1B, TLN1, YWHAQ, YWHAE, YWHAG, YWHAZ.

Techniques: Activation Assay, Translocation Assay, Migration, Transduction, Cell Differentiation

Journal: PLOS One

Article Title: Proteomic profiling of small extracellular vesicles from bovine nucleus pulposus cells

doi: 10.1371/journal.pone.0324179

Figure Lengend Snippet: NP small EV protein cargo and membrane constituents are involved in key metabolic pathways, including glycolysis, gluconeogenesis, Krebs cycle and PPP. They are crucial for energy production and to maintain the cellular redox balance. NP small EVs with the aid of the 20S proteasome, ensure protein quality control and reduced inflammation in a recipient cell. NP small EVs modulate signaling through EPH receptors, impacting cellular communication and tissue organization. Additionally, NP small EV interact with the complement system, influencing the classical, alternative, and lectin pathways involved in immune responses and inflammation. The PI3K/AKT/RAS signaling pathway is axis is impacted by the NP small EV proteome, which could promote ECM synthesis, cell growth and proliferation. Together, these processes underscore the essential role of the NP small EV proteome in sustaining NP cell function and NP niche homeostasis. ECM: Extracellular matrix; EPH: Ephrin receptor, ER: endoplasmic reticulum, ERK: extracellular signal-regulated kinase, MEK: mitogen-activated protein kinase, NP: nucleus pulposus, PDK1: phosphoinositide-dependent protein kinase 1, PPP: pentose phosphate pathway, PI3K/AKT: phosphoinositide 3-kinase/protein kinase B, RAF: rapidly accelerated fibrosarcoma, RHEB: RAS homolog enriched in brain, mTORC1: mammalian target of rapamycin complex 1, small EV: small extracellular vesicles, TSC1/2: tuberous sclerosis proteins 1 and 2. This illustration was created on Biorender. ( www.biorender.com/ ).

Article Snippet: Environmental Information Processing , Regulation of actin cytoskeleton Focal adhesion Rap1 signaling pathway ECM-receptor interaction PI3K-Akt signaling pathway Chemokine signaling pathway Apelin signaling pathway Sphingolipid signaling pathway Ras signaling pathway cAMP signaling pathway MAPK signaling pathway , bta04810 bta04510 bta04015 bta04512 bta04151 bta04062 bta04371 bta04071 bta04014 bta04024 bta04010 , ACTN1, C9, COL6A1, COL6A3, FLNA, FN1, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GSN, HSPA8, HSPB1, HSPG2, ITGA3, ITGAV, ITGB1, LAMC1, MSN, MYH9, PFN1, RAB5B, RAB5C, RAC1, RHOA, RRAS, RRAS2, RAP1B, TLN1, YWHAQ, YWHAE, YWHAG, YWHAZ.

Techniques: Membrane, Control, Cell Function Assay

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Experimental procedure for mice subjected to CRS. D, day. ( B and C ) Representative activity tracking and statistical analysis of time in the center and total distance traveled during OFT ( n = 12 to 13 mice per group). ( D and E ) Representative activity tracking and statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (C). ( F ) Scheme for the analysis of total Rap1 and active Rap1. ( G ) Representative Western blots from the Con and CRS mice. ( H ) Quantification analysis of relative total Rap1 and active Rap1 expression ( n = 5 per group). ( I ) Scheme for delivering vehicle or GGTI-298 into the dHPC and vHPC. ( J ) Representative images of cannula implantation in dHPC and vHPC. ( K and L ) Representative activity tracking and statistical analysis of time in the center and total distance traveled during OFT of mice after administering vehicle and GGTI-298 in dHPC ( n = 8 to 10 mice per group). ( M and N ) Representative activity tracking and statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (L). ( O to R ) Same as in [(K) to (N)] except that the data were from mice receiving vehicle and GGTI-298 in vHPC ( n = 9 to 11 mice per group). Data are presented as the means ± SEM. * P < 0.05 and ** P < 0.01. Two-way ANOVA [(L), (N), (P), and (R)]; Mann-Whitney U test [(C), left]; two-tailed unpaired Student’s t test [(C) right, (E), and (H)].

Article Snippet: C57BL/6J (stock no. N000013, GemPharmatech, China) and Rap1 flx/flx (stock no. 021066, the Jackson Laboratory, US) mice were used in this study.

Techniques: Activity Assay, Western Blot, Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Schematic of AAV injection to express Cre recombinase (Cre-eGFP) or eGFP alone (eGFP) under the CaMKII α promoter in the vHPC of Rap1 flx/flx mice followed by CRS and behavioral tests. ( B ) Representative images showing eGFP expression in vHPC. Scale bar, 500 μm. ( C ) Representative images of CaMKIIα immunostaining in the vCA1. Scale bar, 10 μm. ( D and E ) Representative activity tracking and quantification of time in the center and total distance traveled during OFT ( n = 14 to 15 mice per group). ( F and G ) Representative activity tracking and quantification of time in open arms and open arm entries during EPMT. Same sample size as in [(D) and (E)]. ( H ) Schematic of overexpressing Rap1-DN-mCherry or mCherry alone in the vHPC PNs followed by CRS and behavioral tests. ( I ) Representative images showing mCherry expression in vHPC. Scale bar, 500 μm. ( J ) Representative immunoblots for active Rap1 and total Rap1 and statistical analysis of the immunoblotting assay from the mCherry and Rap1-DN-mCherry mice ( n = 5 per group). ( K to N ) Same as in [(D) to (G)] except that the data were from mice infected with mCherry or Rap1-DN-mCherry ( n = 12 to 15 mice per group). ( O ) Schematic of overexpressing Rap1-CA-mCherry or mCherry alone in the vHPC PNs and subsequent behavioral tests. ( P and Q ) Same as in [(I) and (J)] except that the data were from mice infected with mCherry or Rap1-CA-mCherry ( n = 5 per group). ( R to U ) Same as in [(D) to (G)] except that the data were from mice infected with mCherry or Rap1-CA-mCherry and without subjecting to CRS ( n = 13 to 14 mice per group). Pooled data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Mann-Whitney U test [(E) left, (N) left, and (S) right]; two-tailed unpaired Student’s t test [(E) right, (F), (J), (L), (N) right, (Q), (S) left, and (U)].

Article Snippet: C57BL/6J (stock no. N000013, GemPharmatech, China) and Rap1 flx/flx (stock no. 021066, the Jackson Laboratory, US) mice were used in this study.

Techniques: Injection, Expressing, Immunostaining, Activity Assay, Western Blot, Infection, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Schematics of AAV injections and subsequent CRS and electrophysiological experiments. ( B ) Representative firing traces of vCA1 PNs in response to 200- and 250-pA depolarizing current injections. ( C ) Statistical analysis of AP numbers evoked by different depolarizing current injections ( n = 18 to 19 neurons from five mice per group). ( D ) Representative fAHP traces. ( E ) Statistical analysis of fAHPs. Same sample size as in (C). ( F ) Representative traces showing transient A-type K + currents obtained at various voltages in vCA1 PNs. ( G ) Statistical analysis of transient A-type K + current densities recorded at +40 mV ( n = 12 to 13 neurons from six mice per group). ( H ) Representative blots of the total Kv4.2, Kv4.2 Thr 602 , Kv4.2 Thr 607 , and Kv4.2 Ser 616 in mCherry + CRS and Rap1-DN-mCherry + CRS mice. Statistical analysis of total Kv4.2 ( I ), Kv4.2 Thr 607 ( J ), Kv4.2 Ser 616 ( K ), and Kv4.2 Thr 602 ( L ) expressions in (H) ( n = 3 mice in each group). Data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** p < 0.001. Two-way ANOVA with repeated measures (B); two-tailed unpaired Student’s t test [(E), (G), and (I) to (L)].

Article Snippet: C57BL/6J (stock no. N000013, GemPharmatech, China) and Rap1 flx/flx (stock no. 021066, the Jackson Laboratory, US) mice were used in this study.

Techniques: Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Experimental design for overexpressing Kv4.2-T607A (Kv4.2-T607A) or 3×FLAG alone (vehicle) in CRS mice and subsequent behavioral tests. ( B ) Representative blots and comparisons of the Kv4.2 Thr 607 expression from vehicle and Kv4.2-T607A mice ( n = 3 mice per group). ( C ) Representative traces showing transient A-type K + currents obtained at various voltages in vCA1 PNs and comparisons of transient A-type K + current densities recorded at +40 mV ( n = 7 to 8 neurons from three mice per group). ( D ) Representative activity tracking in OFT. ( E ) Statistical analysis of time in the center and total distance traveled during OFT ( n = 13 to 15 mice per group). ( F ) Representative activity tracking in EPMT. ( G ) Statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (E). ( H ) Representative firing traces of vCA1 PNs in response to depolarizing current injection. ( I ) Statistical analysis of AP numbers evoked by different depolarizing current injections in (H) ( n = 15 to 19 neurons from four to five mice per group). ( J ) Representative fAHP traces. ( K ) Statistical analysis of fAHPs in (J). Same sample size as in (I). ( L ) Schematic depicting the virus injection strategy for inhibiting phosphorylation of Kv4.2 at Thr 607 in Rap1-CA mice and subsequent behavioral tests. ( M to T ) Same as in [(D) to (K)] except that the data were from vehicle + Rap1-CA and Kv4.2-T607A + Rap1-CA mice [(M) to (P), n = 10 to 14 mice per group; (Q) to (T), n = 13 to 15 neurons from four mice per group]. Data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Two-way ANOVA with repeated measures [(I) and (R)]; Mann-Whitney U test [(N) left, and (P) left]; two-tailed unpaired Student’s t test [(B), (C), (E), (G), (K), (N) right, (P) right, and (T)].

Article Snippet: C57BL/6J (stock no. N000013, GemPharmatech, China) and Rap1 flx/flx (stock no. 021066, the Jackson Laboratory, US) mice were used in this study.

Techniques: Expressing, Activity Assay, Injection, Virus, Phospho-proteomics, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: Chronic stress triggers prolonged Rap1 activation in vHPC PNs, facilitating the phosphorylation of Kv4.2 at Thr 607 and diminishing its channel activity. Consequently, when exposed to external anxiogenic environmental cues, the responsiveness of these neurons is heightened, leading to an anxiogenic effect.

Article Snippet: C57BL/6J (stock no. N000013, GemPharmatech, China) and Rap1 flx/flx (stock no. 021066, the Jackson Laboratory, US) mice were used in this study.

Techniques: Activation Assay, Phospho-proteomics, Activity Assay

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Experimental procedure for mice subjected to CRS. D, day. ( B and C ) Representative activity tracking and statistical analysis of time in the center and total distance traveled during OFT ( n = 12 to 13 mice per group). ( D and E ) Representative activity tracking and statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (C). ( F ) Scheme for the analysis of total Rap1 and active Rap1. ( G ) Representative Western blots from the Con and CRS mice. ( H ) Quantification analysis of relative total Rap1 and active Rap1 expression ( n = 5 per group). ( I ) Scheme for delivering vehicle or GGTI-298 into the dHPC and vHPC. ( J ) Representative images of cannula implantation in dHPC and vHPC. ( K and L ) Representative activity tracking and statistical analysis of time in the center and total distance traveled during OFT of mice after administering vehicle and GGTI-298 in dHPC ( n = 8 to 10 mice per group). ( M and N ) Representative activity tracking and statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (L). ( O to R ) Same as in [(K) to (N)] except that the data were from mice receiving vehicle and GGTI-298 in vHPC ( n = 9 to 11 mice per group). Data are presented as the means ± SEM. * P < 0.05 and ** P < 0.01. Two-way ANOVA [(L), (N), (P), and (R)]; Mann-Whitney U test [(C), left]; two-tailed unpaired Student’s t test [(C) right, (E), and (H)].

Article Snippet: Then, the membranes were incubated with the following primary antibodies: Kv4.2 (1:1000, Neuromab, US), phospho-Kv4.2 (1:300, Thr 607 , Santa Cruz, US), phospho-Kv4.2 (1:1000, Ser 616 , MyBioSource, US), phospho-Kv4.2 (1:1000, Thr 602 , Bioss, China), Rap1 (1:1000, Cell Signaling Technology, US), and β-actin (1:2000, Abcam, US), overnight at 4°C.

Techniques: Activity Assay, Western Blot, Expressing, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Schematic of AAV injection to express Cre recombinase (Cre-eGFP) or eGFP alone (eGFP) under the CaMKII α promoter in the vHPC of Rap1 flx/flx mice followed by CRS and behavioral tests. ( B ) Representative images showing eGFP expression in vHPC. Scale bar, 500 μm. ( C ) Representative images of CaMKIIα immunostaining in the vCA1. Scale bar, 10 μm. ( D and E ) Representative activity tracking and quantification of time in the center and total distance traveled during OFT ( n = 14 to 15 mice per group). ( F and G ) Representative activity tracking and quantification of time in open arms and open arm entries during EPMT. Same sample size as in [(D) and (E)]. ( H ) Schematic of overexpressing Rap1-DN-mCherry or mCherry alone in the vHPC PNs followed by CRS and behavioral tests. ( I ) Representative images showing mCherry expression in vHPC. Scale bar, 500 μm. ( J ) Representative immunoblots for active Rap1 and total Rap1 and statistical analysis of the immunoblotting assay from the mCherry and Rap1-DN-mCherry mice ( n = 5 per group). ( K to N ) Same as in [(D) to (G)] except that the data were from mice infected with mCherry or Rap1-DN-mCherry ( n = 12 to 15 mice per group). ( O ) Schematic of overexpressing Rap1-CA-mCherry or mCherry alone in the vHPC PNs and subsequent behavioral tests. ( P and Q ) Same as in [(I) and (J)] except that the data were from mice infected with mCherry or Rap1-CA-mCherry ( n = 5 per group). ( R to U ) Same as in [(D) to (G)] except that the data were from mice infected with mCherry or Rap1-CA-mCherry and without subjecting to CRS ( n = 13 to 14 mice per group). Pooled data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Mann-Whitney U test [(E) left, (N) left, and (S) right]; two-tailed unpaired Student’s t test [(E) right, (F), (J), (L), (N) right, (Q), (S) left, and (U)].

Article Snippet: Then, the membranes were incubated with the following primary antibodies: Kv4.2 (1:1000, Neuromab, US), phospho-Kv4.2 (1:300, Thr 607 , Santa Cruz, US), phospho-Kv4.2 (1:1000, Ser 616 , MyBioSource, US), phospho-Kv4.2 (1:1000, Thr 602 , Bioss, China), Rap1 (1:1000, Cell Signaling Technology, US), and β-actin (1:2000, Abcam, US), overnight at 4°C.

Techniques: Injection, Expressing, Immunostaining, Activity Assay, Western Blot, Infection, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Schematics of AAV injections and subsequent CRS and electrophysiological experiments. ( B ) Representative firing traces of vCA1 PNs in response to 200- and 250-pA depolarizing current injections. ( C ) Statistical analysis of AP numbers evoked by different depolarizing current injections ( n = 18 to 19 neurons from five mice per group). ( D ) Representative fAHP traces. ( E ) Statistical analysis of fAHPs. Same sample size as in (C). ( F ) Representative traces showing transient A-type K + currents obtained at various voltages in vCA1 PNs. ( G ) Statistical analysis of transient A-type K + current densities recorded at +40 mV ( n = 12 to 13 neurons from six mice per group). ( H ) Representative blots of the total Kv4.2, Kv4.2 Thr 602 , Kv4.2 Thr 607 , and Kv4.2 Ser 616 in mCherry + CRS and Rap1-DN-mCherry + CRS mice. Statistical analysis of total Kv4.2 ( I ), Kv4.2 Thr 607 ( J ), Kv4.2 Ser 616 ( K ), and Kv4.2 Thr 602 ( L ) expressions in (H) ( n = 3 mice in each group). Data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** p < 0.001. Two-way ANOVA with repeated measures (B); two-tailed unpaired Student’s t test [(E), (G), and (I) to (L)].

Article Snippet: Then, the membranes were incubated with the following primary antibodies: Kv4.2 (1:1000, Neuromab, US), phospho-Kv4.2 (1:300, Thr 607 , Santa Cruz, US), phospho-Kv4.2 (1:1000, Ser 616 , MyBioSource, US), phospho-Kv4.2 (1:1000, Thr 602 , Bioss, China), Rap1 (1:1000, Cell Signaling Technology, US), and β-actin (1:2000, Abcam, US), overnight at 4°C.

Techniques: Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: ( A ) Experimental design for overexpressing Kv4.2-T607A (Kv4.2-T607A) or 3×FLAG alone (vehicle) in CRS mice and subsequent behavioral tests. ( B ) Representative blots and comparisons of the Kv4.2 Thr 607 expression from vehicle and Kv4.2-T607A mice ( n = 3 mice per group). ( C ) Representative traces showing transient A-type K + currents obtained at various voltages in vCA1 PNs and comparisons of transient A-type K + current densities recorded at +40 mV ( n = 7 to 8 neurons from three mice per group). ( D ) Representative activity tracking in OFT. ( E ) Statistical analysis of time in the center and total distance traveled during OFT ( n = 13 to 15 mice per group). ( F ) Representative activity tracking in EPMT. ( G ) Statistical analysis of time in open arms and open arm entries during EPMT. Same sample size as in (E). ( H ) Representative firing traces of vCA1 PNs in response to depolarizing current injection. ( I ) Statistical analysis of AP numbers evoked by different depolarizing current injections in (H) ( n = 15 to 19 neurons from four to five mice per group). ( J ) Representative fAHP traces. ( K ) Statistical analysis of fAHPs in (J). Same sample size as in (I). ( L ) Schematic depicting the virus injection strategy for inhibiting phosphorylation of Kv4.2 at Thr 607 in Rap1-CA mice and subsequent behavioral tests. ( M to T ) Same as in [(D) to (K)] except that the data were from vehicle + Rap1-CA and Kv4.2-T607A + Rap1-CA mice [(M) to (P), n = 10 to 14 mice per group; (Q) to (T), n = 13 to 15 neurons from four mice per group]. Data are presented as the means ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001. Two-way ANOVA with repeated measures [(I) and (R)]; Mann-Whitney U test [(N) left, and (P) left]; two-tailed unpaired Student’s t test [(B), (C), (E), (G), (K), (N) right, (P) right, and (T)].

Article Snippet: Then, the membranes were incubated with the following primary antibodies: Kv4.2 (1:1000, Neuromab, US), phospho-Kv4.2 (1:300, Thr 607 , Santa Cruz, US), phospho-Kv4.2 (1:1000, Ser 616 , MyBioSource, US), phospho-Kv4.2 (1:1000, Thr 602 , Bioss, China), Rap1 (1:1000, Cell Signaling Technology, US), and β-actin (1:2000, Abcam, US), overnight at 4°C.

Techniques: Expressing, Activity Assay, Injection, Virus, Phospho-proteomics, MANN-WHITNEY, Two Tailed Test

Journal: Science Advances

Article Title: Enhanced Rap1 small GTPase activity in the ventral hippocampus drives stress-induced anxiety

doi: 10.1126/sciadv.adt3163

Figure Lengend Snippet: Chronic stress triggers prolonged Rap1 activation in vHPC PNs, facilitating the phosphorylation of Kv4.2 at Thr 607 and diminishing its channel activity. Consequently, when exposed to external anxiogenic environmental cues, the responsiveness of these neurons is heightened, leading to an anxiogenic effect.

Article Snippet: Then, the membranes were incubated with the following primary antibodies: Kv4.2 (1:1000, Neuromab, US), phospho-Kv4.2 (1:300, Thr 607 , Santa Cruz, US), phospho-Kv4.2 (1:1000, Ser 616 , MyBioSource, US), phospho-Kv4.2 (1:1000, Thr 602 , Bioss, China), Rap1 (1:1000, Cell Signaling Technology, US), and β-actin (1:2000, Abcam, US), overnight at 4°C.

Techniques: Activation Assay, Phospho-proteomics, Activity Assay