Journal: Neuropharmacology

Article Title: HIV gp120 upregulates tonic inhibition through α5-containing GABA A Rs

doi: 10.1016/j.neuropharm.2019.02.024

Figure Lengend Snippet: (A) The p38 antagonist SB203,580 (10 µM) inhibits gp120-induced increases in bicuculline-sensitive current normalized to whole-cell capacitance (two-way ANOVA; F (1,21)=9.14, p=0.006). (B) SB202,474 (10 µM), an inactive analog of SB203580, does not affect the gp120-induced increase in bicuculline-sensitive current (two-way ANOVA; F(1,17)=1.09, p=0.3). (C) RapRp38 expression prevents the gp120-induced increase in tonic inhibition (two-way ANOVA; F(1,27)=5.02, p=0.03). (D) Treatment with rapamycin (200 nM) increases tonic inhibition in RapRp38 expressing cells significantly more than cells expressing tdTomato alone (two-way ANOVA; F(1,22)=6.08, p=0.02). (E) Cycloheximide (CHX) (10 µM) does not affect the gp120-induced increase in tonic inhibition (two-way ANOVA, F(1,24)=0.003, p=0.9). Data are expressed as individual data points with mean ± SEM. Two-way ANOVAs were followed by Tukey’s post hoc test, *p<0.05, **p<0.01, ***p<.001 compared to untreated control. # p<0.05, ##p<0.01, compared to gp120 (A,C) or rapamycin (D) alone (untreated or TdTomato).

Article Snippet: The plasmid encoding RapR-p38 (pCMV5-Flag-RapR-p38) was generated in the laboratory of Klaus Hahn and obtained from Addgene (catalog number: 25935).

Techniques: Expressing, Inhibition, Control

Journal: Neuropharmacology

Article Title: HIV gp120 upregulates tonic inhibition through α5-containing GABA A Rs

doi: 10.1016/j.neuropharm.2019.02.024

Figure Lengend Snippet: Inhibition of CXCR4 with AMD3100 prevents gp120-induced changes in GABAAR signaling. Because gp120IIIB binds to CXCR4 (Islam et al., 2013; Moore et al., 1997) we propose that the signaling cascade starts with this receptor. While multiple cell types including neurons, astrocytes, and microglia express CXCR4, LME treatment to remove microglia prevented gp120-induced changes in GABAAR signaling in neurons. Thus, gp120 does not act directly on neurons to evoke the inhibitory current and microglia are necessary and upstream from the neuronal response. Additionally, because IL-1ra blocked the gp120-induced increase in tonic inhibition and microglia release IL-1β in response to gp120 (Lisi et al., 2011), we placed microglia upstream from the neuron. Finally, p38 signaling in neurons is necessary for the gp120-induced increases in tonic inhibition as shown by RapRp38 expression in neurons. P38 activation is a canonical downstream effector of the IL-1R (Weber et al., 2010), consistent with IL1Rs on neurons mediating the response. P38 activation in other cell types may contribute to upstream steps in the pathways such as IL-1β release from microglia (Schieven, 2005), although these roles were not tested in this study. Activation of p38 increased α5-containing GABAARs on the neuronal cell surface without the need for protein synthesis; the final steps through which p38 regulates GABAAR trafficking are not known. Selective inhibitors are shown in red.

Article Snippet: The plasmid encoding RapR-p38 (pCMV5-Flag-RapR-p38) was generated in the laboratory of Klaus Hahn and obtained from Addgene (catalog number: 25935).

Techniques: Inhibition, Expressing, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels

doi: 10.1128/mcb.01282-09

Figure Lengend Snippet: FIG. 8. Mechanisms of inhibition of IL-2 expression by VIP. (A) Human T cells were cultured with medium (unstim) or CD3/CD28 activated without or with VIP (107 M). After 4 h, nuclear proteins were isolated and assayed for DNA binding to NF- B, AP-1, or NFAT consensus sites by EMSA and expressed as densitometric units relative to the binding of the constitutive nuclear factor NF-Y. The error bars indicate SD. (B) After 3 h, whole-cell extracts were analyzed by Western blotting for c-Jun and phospho-c-Jun expression. NFATp levels were determined in both nuclear and cytoplasmic extracts and expressed as densitometric units relative to laminin B or -tubulin. (C) After 1 h of culture, cell lysates were prepared, and activation of ERK1/2 and MEK1 was determined by Western blotting with phosphospecific Abs and expressed as densitometric units normalized for total ERK1/2 or MEK1. The kinase activity of Raf1 was determined by assaying the phosphotransferase activity toward GST- MEK1. (D) Activation of Rap1 and Ras was determined as described in Materials and Methods. The inactive forms of Raf1 were measured in cell lysates by Western blotting for phospho-Ser43-Raf1 and phospho-Ser259-Raf1. n 3 or 4. *, P 0.01 versus CD3/CD28.

Article Snippet: Protein A/G agarose and antibodies against CD3 chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27kip1, phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Inhibition, Expressing, Cell Culture, Isolation, Binding Assay, Western Blot, Activation Assay, Activity Assay

Journal: Neuropathology and Applied Neurobiology

Article Title: RAPGEF2 mediates oligomeric Aβ‐induced synaptic loss and cognitive dysfunction in the 3xTg‐AD mouse model of Alzheimer’s disease

doi: 10.1111/nan.12686

Figure Lengend Snippet: Activation of the small GTPase Rap2‐JNK pathway in 3xTg‐AD mice. A and B, Cortical lysates from 2.5‐month‐old wild‐type (WT) and 3xTg‐AD (TG) mice were incubated with RalGDS‐RBD agarose beads, followed by immunoblotting for Rap2 (A) or Rap1 (B), respectively. C and D, Quantification of active Rap2 (C) and active Rap1 (D) levels normalised to total Rap2 and Rap1 respectively ( n = 9; 6 males, 3 females). E and F, Cortical lysates from 2.5‐month‐old wild‐type (WT) and 3xTg‐AD (TG) mice were collected and subjected to western blotting for activation (phosphorylation) of JNK (E) and p38 (F). G and H, Quantification of pJNK (G, n = 6; 5 males, 1 female) and p‐p38 (H, n = 6; 5 males, 1 female) levels normalised to total expression. All data are shown as the mean ± SEM. * p < 0.05, *** p < 0.001; two‐tailed unpaired Student's t ‐test

Article Snippet: Antibodies against phospho‐p38 (#4511), p38 (#9212), phospho‐JNK (#9255), JNK (#9252), Rap1 (#2399) and β‐tubulin (#2146) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Incubation, Western Blot, Phospho-proteomics, Expressing, Two Tailed Test

Journal: Frontiers in bioscience (Landmark edition)

Article Title: F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

doi: 10.31083/j.fbl2905177

Figure Lengend Snippet: Fig. 9. F2RL3 regulates the expression of Rap1/MAPK signaling pathway in GC cells. (A) Expression of proteins related to F2RL3

Article Snippet: The protein content in the sample was quantified using the bicinchoninic acid assay, followed by electrophoresis, electrotransfer of proteins to a membrane, and incubation with the following primary antibodies: F2RL3 (#ab137927; Abcam, Cambridge, MA, USA), E-cadherin (#ab40772; Abcam), vimentin (#ab92547; Abcam), Rap1 GTPaseactivating-protein (Rap1GAP) (#ab32373; Abcam), Rap1 (#ab175329; Abcam), phosphorylated extracellular signalregulated kinase 1/2 (p-ERK1/2) (#4370T; Cell Signaling Technology [CST], Danvers, MA, USA), ERK1/2 (#9194S; CST), p-p38 MAPK (#4511S; CST), p38 MAPK (#8690S; CST), and Snail (#3879S; CST). β-actin (#93473SF; CST) was selected as the internal reference gene.

Techniques: Expressing

Journal: Frontiers in bioscience (Landmark edition)

Article Title: F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

doi: 10.31083/j.fbl2905177

Figure Lengend Snippet: Fig. 10. Effects of the Rap1/MAPK signaling pathway on the EMT of GC cells. (A) Expression of F2RL3, E-cadherin, vimentin, and Snail detected by Western blotting. (B) Bar chart showing the expression of F2RL3, vimentin, E-cadherin, and Snail. (C) Activity

Article Snippet: The protein content in the sample was quantified using the bicinchoninic acid assay, followed by electrophoresis, electrotransfer of proteins to a membrane, and incubation with the following primary antibodies: F2RL3 (#ab137927; Abcam, Cambridge, MA, USA), E-cadherin (#ab40772; Abcam), vimentin (#ab92547; Abcam), Rap1 GTPaseactivating-protein (Rap1GAP) (#ab32373; Abcam), Rap1 (#ab175329; Abcam), phosphorylated extracellular signalregulated kinase 1/2 (p-ERK1/2) (#4370T; Cell Signaling Technology [CST], Danvers, MA, USA), ERK1/2 (#9194S; CST), p-p38 MAPK (#4511S; CST), p38 MAPK (#8690S; CST), and Snail (#3879S; CST). β-actin (#93473SF; CST) was selected as the internal reference gene.

Techniques: Expressing, Western Blot, Activity Assay

Journal: Frontiers in bioscience (Landmark edition)

Article Title: F2RL3 Regulates Epithelial-Mesenchymal Transition and Angiogenesis in Gastric Cancer through the Rap1/MAPK Signaling Pathway.

doi: 10.31083/j.fbl2905177

Figure Lengend Snippet: Fig. 11. Impact of the Rap1/MAPK signaling pathway on angiogenesis. (A) Activity of HUVECs detected by the CCK-8. (B)

Article Snippet: The protein content in the sample was quantified using the bicinchoninic acid assay, followed by electrophoresis, electrotransfer of proteins to a membrane, and incubation with the following primary antibodies: F2RL3 (#ab137927; Abcam, Cambridge, MA, USA), E-cadherin (#ab40772; Abcam), vimentin (#ab92547; Abcam), Rap1 GTPaseactivating-protein (Rap1GAP) (#ab32373; Abcam), Rap1 (#ab175329; Abcam), phosphorylated extracellular signalregulated kinase 1/2 (p-ERK1/2) (#4370T; Cell Signaling Technology [CST], Danvers, MA, USA), ERK1/2 (#9194S; CST), p-p38 MAPK (#4511S; CST), p38 MAPK (#8690S; CST), and Snail (#3879S; CST). β-actin (#93473SF; CST) was selected as the internal reference gene.

Techniques: Activity Assay, CCK-8 Assay

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Establishment of the risk signature with MAPK-RAP1A related genes in the training (TCGA) and validation database (ICGC). (A) RT-qPCR was used to detect the expression of ERK, JNK, p38, RAP1A in 20 cases HCC tumor tissues and adjacent tissues. (B) Western blot was used to detect the expression of ERK, JNK, p38, RAP1A in Huh7, HepG2, Hepa1-6, and L02 cell lines. (C) Multivariate Cox regression analysis to validate prognosis related clinicopathological characters. (D) The correlation of our MAPK-RAP1A risk signature with the clinicopathological characters (TNM stage) of HCC from the training set. (E) ROC curves of AUC evaluated the efficiency of the risk signature for predicting TNM stage in training set. (F) Multivariate Cox regression analysis to validate prognosis related clinicopathological characters. (G) Nomogram for predicting 1-, 3-, and 5-year OS of LIHC patients in the ICGC cohort. * p < 0.05; *** p < 0.001.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Screening out genes for constructing a risk signature. (A) Log (Lambda) value of the 11 genes in LASSO model. (B) The most proper log (Lambda) value in LASSO model. (C) Six genes (STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF) were selected for constructing a risk signature using multivariate Cox regression model. (D) A nomogram for predicting 1-, 3- and 5-year survival rate of HCC patients was established. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques:

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: The risk signature in HCC from the training set, the independent external validation cohort. (A, C) The Kaplan–Meier curves of the MAPK-RAP1A related risk signature. (B, D) Time-dependent ROC curves at 1, 3, and 5 years. AUC, area under the ROC; HCC, hepatocellular carcinoma; ROC, receiver-operating characteristic.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Biomarker Discovery

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Validation of signature genes in the MAPK-RAP1A dataset. (A) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different TNM stages. (B) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different T stages. (C) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different N stages. (D) Expression of STMN1, FLT3, ANGPT2, HSPA8, RAP1A, and PGF in HCC samples with different M stages.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Biomarker Discovery, Expressing

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Establishment of the MAPK-RAP1A with immunity in the TCGA database. (A, B) PCA based on MAPK-RAP1A gene signature between low-immunity group and high-immunity group. (C) Tumor purity in different immunity subgroups. (D) Kaplan–Meier curves of overall survival according to immunity groups in the training cohort.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques:

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Validation of immune estimate in the MAPK-RAP1A dataset. (A) The corresponding stromal score, immune score, and estimated score of MAPK-RAP1A gene signature between low-immunity group and high-immunity group. (B) Scores of stromal, immune, and ESTIMATE in HCC samples with different TNM stages. (C) Scores of stromal, immune, and ESTIMATE in HCC samples with different T stages. (D) Scores of stromal, immune, and ESTIMATE in HCC samples with different N stages. (E) Scores of stromal, immune, and ESTIMATE in HCC samples with different M stages.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Biomarker Discovery

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Immune infiltration construction and validation in HCC. (A) Plot for comparison of the immune cell fraction difference between high risk and low risk. Fractions of each immune cell type were compared. Association of hub genes’ expression with immune infiltration in HCC. (B) PGF (C) STMN1, (D) ANGPT2, (E) FLT3, (F) HSPA8, and (G) RAP1A. P < 0.05 denotes significance. Each dot represents a sample in the TCGA-LIHC dataset.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Biomarker Discovery, Comparison, Expressing

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of hub genes in the TCGA-LIHC MAPK-RAP1A dataset. (A–F) Top three gene sets (according to GSEA enrichment score) enriched in the high-expression group of single hub genes. (A) PGF, (B) STMN1, (C) ANGPT2, (D) FLT3, (E) HSPA8, and (F) RAP1A.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: MAPK-RAP1A Signaling Enriched in Hepatocellular Carcinoma Is Associated With Favorable Tumor-Infiltrating Immune Cells and Clinical Prognosis

doi: 10.3389/fonc.2021.649980

Figure Lengend Snippet: Effect of the risk signature and immune checkpoint gene expression on patient survival. (A) Association between the risk signature and immune checkpoint gene expression. (B) Association between the hub gene and immune checkpoint gene expression. (C) Immunohistochemical staining was used to compare the expression of PGF, STMN1, ANGPT2, FLT3, RAP1A, HSPA8 with B7 in HCC tissues. (D) Kaplan–Meier survival curves of OS among patient groups stratified by the risk signature and immune checkpoint gene expression. (E) Association between B7 (CD80) and PDL1 in HCC tissues compared with adjacent tissues. Supplemental Figure Immunohistochemistry was used to detect the negative control, high and low expression of PGF, STMN1, ANGPT2, FLT3, RAP1A, HSPA8, and B7 in HCC tissues at high and low magnification.

Article Snippet: Then incubated in 5% Bovine Serum Albumin at room temperature for 2 h. The membranes were exposed to primary antibodies against ERK, p-ERK, JNK, p-JNK, p38, p-p38, and RAP1A (1:1,000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or β -actin (1:1,000 dilution; Cell Signaling Technology, USA) at 4°C overnight.

Techniques: Gene Expression, Immunohistochemical staining, Staining, Expressing, Immunohistochemistry, Negative Control

Journal: Journal of Biological Chemistry

Article Title: Cyclic AMP Promotes cAMP-responsive Element-binding Protein-dependent Induction of Cellular Inhibitor of Apoptosis Protein-2 and Suppresses Apoptosis of Colon Cancer Cells through ERK1/2 and p38 MAPK

doi: 10.1074/jbc.m313346200

Figure Lengend Snippet: FIG. 3. CREB phosphorylation is mediated by ERK1/2 and p38 MAPK. T84 cells were serum-starved for 12 h and incubated for 1 h with SB202190 (SB, 20 M), U0126 (U, 10 M), and (Rp)-cAMPS (Rp, 100 M) alone or in combination as indicated. After 30 min of further incubation with 8-CPT-cAMP (CPT, 100 M), cells were lysed in SDS sample buffer, and proteins were analyzed by immunoblotting with the indicated antibodies. The lower graph shows densitometric results of the blots for phospho-CREB normalized to total CREB expression. Data are expressed relative to unstimulated controls without inhibitors and are mean S.E. (n 3; *, p 0.05; N.S., not significant). Inhibitors, with the exception of (Rp)-cAMPS alone (lane b), significantly decreased CREB phosphorylation (lanes c–f, p 0.05). IB, immunoblot; pAb, polyclonal antibody.

Article Snippet: Polyclonal antibodies against CREB, phospho-CREB, phospho-MSK1, phospho-p90RSK, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, and phospho-p38 MAPK and the MEK1/2 inhibitors PD98059 and U0126 were obtained from Cell Signaling Technology Inc. (San Diego, CA). mAb against Rap1 was from BD Bioscience Pharmingen.

Techniques: Phospho-proteomics, Incubation, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Cyclic AMP Promotes cAMP-responsive Element-binding Protein-dependent Induction of Cellular Inhibitor of Apoptosis Protein-2 and Suppresses Apoptosis of Colon Cancer Cells through ERK1/2 and p38 MAPK

doi: 10.1074/jbc.m313346200

Figure Lengend Snippet: FIG. 4. cAMP activates ERK1/2 and p38 MAPK in a PKA-independent manner. A, T84 cells were serum-starved for 12 h and treated with PGE2 (1 M), Fsk (10 M), 8-CPT-cAMP (CPT, 100 M), or TNF (100 ng/ml) for 30 min. Cells were lysed and analyzed by immunoblotting with the indicated antibodies. B and C, cells, serum-starved for 12 h, were treated with H89 (10 M), (Rp)-cAMPS (Rp, 100 M), or U0126 (U, 10 M) for 1 h and incubated with 8-CPT-cAMP (CPT, 100 M) for 30 min. Cell lysates were prepared and analyzed by non-radioactive kinase assay (KA). Immunoprecipitated phospho-ERK1/2 or phospho-p38 MAPK were incubated with Elk or ATF-2 fusion protein for 30 min at 30 °C in the presence of ATP (200 M), and phosphorylated fusion proteins were analyzed by immunoblotting with the indicated antibodies (KA, top panel). The total amount of fusion protein present in each reaction was visualized by Coomassie Blue staining (KA, second panel). Total cell lysates were analyzed in parallel by immunoblotting (IB) with the indicated antibodies (third and fourth panels). D and E, T84 cells (D), serum-starved for 12 h, or CHO cells (E) were treated with H89 (10 M) for 1 h and incubated with 8-CPT-cAMP (CPT, 100 M) or 8-CPT-2-O-Me-cAMP (Me, 100 M) for the indicated times (D) or 15 min (E). Cells were lysed and analyzed by immunoblotting with indicated antibodies. F, T84 cells, serum-starved for 12 h, were incubated with 8-CPT-cAMP (CPT, 100 M), 8-CPT-2-O-Me-cAMP (Me, 100 M), or phorbol 12-myristate 13-acetate (PMA, 1 M) for 2 min. Cells were lysed and assayed for Rap1 activation with a pull-down assay. Precipitates (top panel) and total cell lysates (bottom panel) were analyzed by immunoblotting with anti-Rap1 mAb. pAb, polyclonal antibody.

Article Snippet: Polyclonal antibodies against CREB, phospho-CREB, phospho-MSK1, phospho-p90RSK, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, and phospho-p38 MAPK and the MEK1/2 inhibitors PD98059 and U0126 were obtained from Cell Signaling Technology Inc. (San Diego, CA). mAb against Rap1 was from BD Bioscience Pharmingen.

Techniques: Western Blot, Incubation, Kinase Assay, Immunoprecipitation, Staining, Activation Assay, Pull Down Assay

Journal: Journal of Biological Chemistry

Article Title: Cyclic AMP Promotes cAMP-responsive Element-binding Protein-dependent Induction of Cellular Inhibitor of Apoptosis Protein-2 and Suppresses Apoptosis of Colon Cancer Cells through ERK1/2 and p38 MAPK

doi: 10.1074/jbc.m313346200

Figure Lengend Snippet: FIG. 5. cAMP induces c-IAP2 protein expression through ERK1/2 and p38 MAPK activation. A, T84 cells, transfected with pGL3-c-IAP2-Luc and pSV40-RL-Luc, were treated for 1 h with (Rp)- cAMPS (Rp, 100 M), SB202910 (SB, 20 M), and U0126 (U, 10 M) alone or in combination and stimulated for 12 h with CTX (250 ng/ml) or 6 h with 8-CPT-cAMP (CPT, 100 M). Cell extracts were assayed by a dual luciferase assay. Results represent relative promoter activity compared with CTX- or CPT-treated cells without inhibitors as 100% and are mean S.E. (n 3; *, p 0.05 versus CTX- or CPT-treated cells without inhibitors; #, p 0.05 relative to cells treated with either of the inhibitors alone; N.S., not significant). B, T84 cells were treated for 1 h with (Rp)-cAMPS (Rp, 100 M), SB202910 (SB, 20 M), and/or U0126 (U, 10 M) as indicated and incubated for 12 h with CTX (250 ng/ml) or 6 h with 8-CPT-cAMP (CPT, 100 M). Cells were lysed in SDS sample buffer, and proteins were analyzed by immunoblotting with the indicated antibodies. The lower graphs show densitometric results of the respective blots normalized against actin levels and expressed rel- ative to unstimulated controls without inhibitors and are mean S.E. (n 3; *, p 0.05 versus untreated controls; #, p 0.05; N.S., not significant). C, T84 cells were transfected with pUSE-MEK1 (S218D/ S222D) (MEK1-active) and/or pBabeHygro-MKK3A (MKK3-active), pGL3-c-IAP2-Luc, and pSV40-RL-Luc and incubated for 12 h. Cell extracts were assayed by a dual luciferase assay. Results represent relative increases compared with vector-transfected controls and are mean S.E. (n 3; *, p 0.05 versus vector-transfected cells; #, p 0.05). IB, immunoblot; pAb, polyclonal antibody.

Article Snippet: Polyclonal antibodies against CREB, phospho-CREB, phospho-MSK1, phospho-p90RSK, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, and phospho-p38 MAPK and the MEK1/2 inhibitors PD98059 and U0126 were obtained from Cell Signaling Technology Inc. (San Diego, CA). mAb against Rap1 was from BD Bioscience Pharmingen.

Techniques: Expressing, Activation Assay, Transfection, Luciferase, Activity Assay, Incubation, Western Blot, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Cyclic AMP Promotes cAMP-responsive Element-binding Protein-dependent Induction of Cellular Inhibitor of Apoptosis Protein-2 and Suppresses Apoptosis of Colon Cancer Cells through ERK1/2 and p38 MAPK

doi: 10.1074/jbc.m313346200

Figure Lengend Snippet: FIG. 6. cAMP regulates apoptosis through ERK1/2 and p38 MAPK. T84 cells were incubated for 1 h with H89 (10 M), (Rp)-cAMPS (Rp, 100 M), SB202910 (SB, 20 M), and/or U0126 (U, 10 M) as indicated followed by further incubation for 1 h with CTX (250 ng/ml) or 8-CPT-cAMP (CPT, 100 M). Cells were stimulated with staurosporine (200 nM) for 12 h, and oligonucleosome release into the cytoplasm was assayed by enzyme-linked immunosorbent assay (A and B), or for 6 h, and caspase-3 activity was assayed by enzyme-linked immunosorbent assay (C). A and C, results represent relative increases compared with untreated cells without inhibitors, respectively, and are mean S.E. (n 3; *, p 0.05 versus staurosporine-treated cells without CTX or CPT stimulation; #, p 0.05). B, results represent relative levels of 8-CPT-cAMP-mediated inhibition of DNA fragmentation induced by staurosporine compared with CPT- and staurosporine-treated cells without inhibitors as 100% and are shown as mean S.E. (n 3; *, p 0.05 versus CPT- and staurosporine-treated cells without inhibitors).

Article Snippet: Polyclonal antibodies against CREB, phospho-CREB, phospho-MSK1, phospho-p90RSK, p44/42 MAPK, phospho-p44/42 MAPK, p38 MAPK, and phospho-p38 MAPK and the MEK1/2 inhibitors PD98059 and U0126 were obtained from Cell Signaling Technology Inc. (San Diego, CA). mAb against Rap1 was from BD Bioscience Pharmingen.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Activity Assay, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

doi: 10.1128/MCB.01282-09

Figure Lengend Snippet: VIP-induced suppressive T cells are arrested at the early G1 phase of the cell cycle. (A) Effects of VIP on the expression of G1 cyclins and CDK and CDKi proteins. Human T cells were CD3/CD28 activated without or with VIP (10−7 M) for different time intervals. The cell lysates were subjected to Western blot analysis for cyclins, CDKs, p27kip1, phosphorylated pRb (ppRb), or actin. (B) IL-2 partially reverses VIP effects on cyclins and p27kip1. Human T cells were CD3/CD28 activated without or with VIP (10−7 M) and IL-2 (50 U/ml) for 72 h. Western blot analysis was performed on cell lysates for cyclins, p27kip1, and Zap70. (C) VIP inhibits cyclin/CDK activity. Human T cells were CD3/CD28 activated without or with VIP (10−7 M) for 36 h. The cell lysates were immunoprecipitated (IP) with Abs against cyclin D2, cyclin D3, cyclin E, cdk2, or cdk4. Cyclin D2-cdk4, cyclin D3-cdk4, and cyclin E-cdk2 interactions were determined by Western blot (wb) analysis of precipitates with anti-cyclin D2, anti-cyclin D3, and anti-cdk2, respectively. cdk4-cyclin D2/D3 and cyclin E-cdk2 kinase activities were measured using pRb-GST and histone H1, respectively, as substrates. n = 3. (See Fig. S5 in the supplemental material for quantitative analysis.) unstim, unstimulated.

Article Snippet: Protein A/G agarose and antibodies against CD3ζ chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27 kip1 , phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Activity Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

doi: 10.1128/MCB.01282-09

Figure Lengend Snippet: VIP maintains threshold levels of p27kip1. Human T cells were CD3/CD28 activated without or with VIP (10−7M). (A) VIP increases the interaction of p27kip1 with cyclin/CDK complexes. Cell lysates obtained after 24 h of culture were immunoprecipitated (IP) with Abs against cyclin D2, cyclin E, cdk2, or cdk4 and analyzed by Western blotting (wb) for cyclins, CDK, and p27kip1 (see Fig. S6A in the supplemental material for quantitative data). (B) VIP inhibits p27kip1 phosphorylation in activated T cells. After 48 h of culture, the cell lysates were immunoprecipitated with anti-p27kip1 Abs and analyzed by Western blotting for p27kip1 (total p27kip1) or phospho-Thr (phosphorylated p27kip1 [P-p27kip1]) and expressed as densitometric units relative to unstimulated samples. The error bars indicate SD. (C) VIP increases p27kip1 mRNA levels. p27kip1 mRNA expression was determined by Northern blotting and expressed as densitometric units relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When indicated, actinomycin D (ActD) was added during culture (the blot corresponds to 12 h). *, P < 0.001 versus CD3/CD28 (n = 3).

Article Snippet: Protein A/G agarose and antibodies against CD3ζ chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27 kip1 , phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics, Expressing, Northern Blot

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

doi: 10.1128/MCB.01282-09

Figure Lengend Snippet: VIP inhibits FOXO1 phosphorylation and the Akt/PI3K pathway. Human T cells were cultured with medium (unstim) or CD3/CD28 activated in the absence or presence of VIP (10−7 M). (A) VIP inhibits phosphorylation and cytoplasmic translocation of FOXO1. Total cell extracts isolated at 4 h of culture (left) or nuclear and cytoplasmic protein extracts isolated at 8 h of culture (right) were analyzed by Western blotting for FOXO1 or phospho-FOXO1 and expressed as densitometric units relative to total FOXO1, laminin B, or β-tubulin. The error bars indicate SD. (B) VIP inhibits Akt/PKB phosphorylation and kinase activity in stimulated T cells. Cell lysates isolated after 2 h of culture were analyzed by Western blotting for phospho-Akt and PDK1 and expressed as densitometric units relative to total Akt or PDK1. Akt kinase activity was determined as described in Materials and Methods and expressed as percentages of the Akt activity of CD3/CD28-activated T cells. (C) The VIP effect on Akt activity is dependent on PI3K and PP2A activities. CD3/CD28-stimulated human T cells were treated with VIP and/or the PI3K activators peroxovanadate (PV) (50 μM) and 740 Y-P (20 μM) or the PP2A inhibitor okadaic acid (OA) (1 μM). Western blot analysis was performed on cell lysates isolated 2 h (for phospho-Akt), 4 h (for phospho-FOXO1), or 48 h (for p27kip1) of culture. The results are expressed as densitometric units relative to total Akt or β-tubulin. (D) Involvement of inhibition of PI3K-Akt signaling in the VIP effect on the T-cell cycle and regulatory functions. CD3/CD28-activated human T cells were treated with VIP and/or PV, OA, or 740 Y-P. The percentage of cells in S phase and proliferation were determined after 72 h. After 96 h in culture, T cells were recovered, and their suppressive potential was determined in cocultures with syngeneic responder T cells (at a suppressor/responder ratio of 1:2) and allogeneic mDCs, as described in the legend to Fig. ​Fig.3A,3A, and expressed as a percentage of inhibition of proliferation. n = 3 or 4. *, P < 0.01 versus CD3/CD28; #, P < 0.01 versus VIP-treated samples.

Article Snippet: Protein A/G agarose and antibodies against CD3ζ chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27 kip1 , phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Cell Culture, Translocation Assay, Isolation, Western Blot, Activity Assay, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels ▿ †

doi: 10.1128/MCB.01282-09

Figure Lengend Snippet: VIP mediates the suppressive effect by elevating cAMP levels. (A) VIP increases cAMP levels and stimulates PKA activity in human activated T cells. Human T cells were CD3/CD28 stimulated with or without VIP (10−7 M). Intracellular cAMP levels were determined at different times (top). PKA activity was assayed by radioimmunoassay in cell lysates obtained 1 h after stimulation (bottom). The error bars indicate SD. (B to E) Human T cells were CD3/CD28 stimulated in the absence (−) or presence of forskolin (10−6 M), 8-Br-cAMP (0.1 mM), or VIP (10−7 M) with or without H89 (50 ng/ml) or Rp-cAMP (0.1 mM). (B) Cell cycle analysis was performed after 72 h. (C) Expression of the soluble and membrane forms of CTLA4 was determined at 96 h. (D) The suppressive activity on effector T cells was determined as described in the legend to Fig. ​Fig.3A3A at an effector/suppressor cell ratio of 2:1. (E) The expression and phosphorylation statuses of cyclins, CDK, p27kip1, FOXO1, Akt, Raf1, and ERK1/2 and the activation of cyclin-CDK complexes and Ras were quantified as detailed in the legends to Fig. ​Fig.55 to ​to8.8. The specificity of H89 and Rp-cAMP in our study was confirmed by their blocking effects on the actions of forskolin and Br-cAMP. Representative blots are shown in Fig. S9 in the supplemental material. n = 3 or 4. *, P < 0.001 versus CD3/CD28; #, P < 0.001 versus VIP-treated samples.

Article Snippet: Protein A/G agarose and antibodies against CD3ζ chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27 kip1 , phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Activity Assay, RIA Assay, Cell Cycle Assay, Expressing, Membrane, Phospho-proteomics, Activation Assay, Blocking Assay

Journal: Molecular Medicine Reports

Article Title: Saikosaponin D inhibits autophagosome-lysosome fusion and induces autophagy-independent apoptosis in MDA-MB-231 breast cancer cells

doi: 10.3892/mmr.2020.11155

Figure Lengend Snippet: SSD activates the p38 mitogen-activated protein kinase signaling pathway. (A) MDA-MB-231 cells were exposed to SSD for 24 h and the expression levels of JNK, p-JNK, ERK, p-ERK, p38 and p-p38 were detected by western blotting (n=3). (B) Semi-quantitative analysis of the expression levels from part A. *P<0.05, **P<0.01 vs. 0 µM. MDA-MB-231 cells were pretreated with 10 µM SB203580 for 2 h and then exposed to 10 µM SSD for 24 h. (C) Apoptotic cells were determined using flow cytometry (n=3). (D) Quantitative analysis of the apoptotic rate. **P<0.01. (E) Expression levels of c-caspase-3 and c-PARP were detected using western blotting. (F) Semi-quantitative analysis of the expression levels in part E. **P<0.01 vs. control. (G) Cell viability was determined using MTT assays (n=3). *P<0.05. Data are presented as the mean ± SD. SSD, saikosaponin D; c-, cleaved; p-, phosphorylated; PI, propidium iodide; PARP, poly (ADP-ribose) polymerase.

Article Snippet: The following reagents were used in the present study: SSD (cat. no. A0259; Chengdu Must Bio-Technology Co. Ltd.), bafilomycin A1 (Baf; cat. no. 11038; Cayman Chemical Company), rapamycin (Rapa; cat. no. S1039; Selleck Chemicals), SB230580 (p38 MAPK inhibitor; cat. no. S1076; Selleck Chemicals), LysoTrackerTM Red DND-99 (cat. no. L7528; Thermo Fisher Scientific, Inc.) DAPI (cat. no. C1006; Beyotime Institute of Biotechnology) and DMSO (as solvent and placebo; cat. no. D2650; Sigma-Aldrich; Merck KGaA).

Techniques: Expressing, Western Blot, Flow Cytometry, Control

Journal: Molecular Medicine Reports

Article Title: Saikosaponin D inhibits autophagosome-lysosome fusion and induces autophagy-independent apoptosis in MDA-MB-231 breast cancer cells

doi: 10.3892/mmr.2020.11155

Figure Lengend Snippet: Excessive accumulation of autophagosomes does not contribute to SSD-mediated apoptosis. Stable knockdown MDA-MB-231 cells (shCON or shATG5) were exposed to 10 µM SSD for 24 h. (A) Following treatment, the expression levels of ATG5, LC3B-II, p-p38, p38, c-caspase-3 and c-PARP were detected using western blotting. (B) Semi-quantitative analysis of the expression levels in part A, n=3; **P<0.01. (C) Apoptotic cells were determined using flow cytometry. (D) Quantitative analysis of the apoptotic rate form part C. n=3. (E) Cell viability was determined using MTT assays. n=3. All data are presented as the mean ± SD. sh, small hairpin RNA; CON, control; ATG, autophagy-related protein; c-, cleaved; n.s., not significant; p-, phosphorylated; LC3B, microtubule-associated protein 1 light chain 3 β; PARP, poly (ADP-ribose) polymerase; SSD, saikosaponin D; PI, propidium iodide.

Article Snippet: The following reagents were used in the present study: SSD (cat. no. A0259; Chengdu Must Bio-Technology Co. Ltd.), bafilomycin A1 (Baf; cat. no. 11038; Cayman Chemical Company), rapamycin (Rapa; cat. no. S1039; Selleck Chemicals), SB230580 (p38 MAPK inhibitor; cat. no. S1076; Selleck Chemicals), LysoTrackerTM Red DND-99 (cat. no. L7528; Thermo Fisher Scientific, Inc.) DAPI (cat. no. C1006; Beyotime Institute of Biotechnology) and DMSO (as solvent and placebo; cat. no. D2650; Sigma-Aldrich; Merck KGaA).

Techniques: Knockdown, Expressing, Western Blot, Flow Cytometry, Control

Journal: Frontiers in Pharmacology

Article Title: Swainsonine Triggers Paraptosis via ER Stress and MAPK Signaling Pathway in Rat Primary Renal Tubular Epithelial Cells

doi: 10.3389/fphar.2021.715285

Figure Lengend Snippet: SW increases the expression of proteins involved in the MAPK cascade in RTECs (A) The proteins levels changes of ERK, JNK and p38 were analyzed by the Western blotting analysis of cells treated with a range of concentration of the SW for 24 h (B–D) These column charts show the ratio of p-ERK/ERK, p-JNK/JNK, p-p38/p38. Three independent experiments were performed. Statistically significance was indicated: * p < 0.05, ** p < 0.01 compared with control.

Article Snippet: Bafilomycin A1 (Baf A1) (HY-100558), Rapamycin (Rapa) (HY-10219), U0126 (HY-12031), SP600125 (HY-12041) and 4-Phenylbutyric acid (4-PBA) (HY-A0281) were obtained from MCE, United States p-PERK (#3179, Rabbit, anti-rat), ATF6 (#65880, Rabbit, anti-rat), p38 (#8690, Rabbit, anti-rat), and p-p38 (#4092, Rabbit, anti-rat) antibodies were purchased from Cell Signaling Technology, United States.

Techniques: Expressing, Western Blot, Concentration Assay, Control

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels

doi: 10.1128/mcb.01282-09

Figure Lengend Snippet: FIG. 5. VIP-induced suppressive T cells are arrested at the early G1 phase of the cell cycle. (A) Effects of VIP on the expression of G1 cyclins and CDK and CDKi proteins. Human T cells were CD3/CD28 activated without or with VIP (107 M) for different time intervals. The cell lysates were subjected to Western blot analysis for cyclins, CDKs, p27kip1, phosphorylated pRb (ppRb), or actin. (B) IL-2 partially reverses VIP effects on cyclins and p27kip1. Human T cells were CD3/CD28 activated without or with VIP (107 M) and IL-2 (50 U/ml) for 72 h. Western blot analysis was performed on cell lysates for cyclins, p27kip1, and Zap70. (C) VIP inhibits cyclin/CDK activity. Human T cells were CD3/CD28 activated without or with VIP (107 M) for 36 h. The cell lysates were immunoprecipitated (IP) with Abs against cyclin D2, cyclin D3, cyclin E, cdk2, or cdk4. Cyclin D2-cdk4, cyclin D3-cdk4, and cyclin E-cdk2 interactions were determined by Western blot (wb) analysis of precipitates with anti-cyclin D2, anti-cyclin D3, and anti-cdk2, respectively. cdk4-cyclin D2/D3 and cyclin E-cdk2 kinase activities were measured using pRb-GST and histone H1, respectively, as substrates. n 3. (See Fig. S5 in the supplemental material for quantitative analysis.) unstim, unstimulated.

Article Snippet: Protein A/G agarose and antibodies against CD3 chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27kip1, phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Activity Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels

doi: 10.1128/mcb.01282-09

Figure Lengend Snippet: FIG. 6. VIP maintains threshold levels of p27kip1. Human T cells were CD3/CD28 activated without or with VIP (107M). (A) VIP increases the interaction of p27kip1 with cyclin/CDK complexes. Cell lysates obtained after 24 h of culture were immunoprecipitated (IP) with Abs against cyclin D2, cyclin E, cdk2, or cdk4 and analyzed by Western blotting (wb) for cyclins, CDK, and p27kip1 (see Fig. S6A in the supplemental material for quantitative data). (B) VIP inhibits p27kip1 phosphorylation in activated T cells. After 48 h of culture, the cell lysates were immunoprecipitated with anti-p27kip1 Abs and analyzed by Western blotting for p27kip1 (total p27kip1) or phospho-Thr (phosphorylated p27kip1 [P-p27kip1]) and expressed as densitometric units relative to unstimulated samples. The error bars indicate SD. (C) VIP increases p27kip1 mRNA levels. p27kip1

Article Snippet: Protein A/G agarose and antibodies against CD3 chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27kip1, phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Phospho-proteomics

Journal: Molecular and Cellular Biology

Article Title: Vasoactive Intestinal Peptide Induces Cell Cycle Arrest and Regulatory Functions in Human T Cells at Multiple Levels

doi: 10.1128/mcb.01282-09

Figure Lengend Snippet: FIG. 9. VIP mediates the suppressive effect by elevating cAMP levels. (A) VIP increases cAMP levels and stimulates PKA activity in human activated T cells. Human T cells were CD3/CD28 stimulated with or without VIP (107 M). Intracellular cAMP levels were determined at different times (top). PKA activity was assayed by radioimmunoassay in cell lysates obtained 1 h after stimulation (bottom). The error bars indicate SD. (B to E) Human T cells were CD3/CD28 stimulated in the absence () or presence of forskolin (106 M), 8-Br-cAMP (0.1 mM), or VIP (107 M) with or without H89 (50 ng/ml) or Rp-cAMP (0.1 mM). (B) Cell cycle analysis was performed after 72 h. (C) Expression of the soluble and membrane forms of CTLA4 was determined at 96 h. (D) The suppressive activity on effector T cells was determined as described in the legend to Fig. 3A at an effector/suppressor cell ratio of 2:1. (E) The expression and phosphorylation statuses of cyclins, CDK, p27kip1, FOXO1, Akt, Raf1, and ERK1/2 and the activation of cyclin-CDK complexes and Ras were quantified as detailed in the legends to Fig. 5 to 8. The specificity of H89 and Rp-cAMP in our study was confirmed by their blocking effects on the actions of forskolin and Br-cAMP. Representative blots are shown in Fig. S9 in the supplemental material. n 3 or 4. *, P 0.001 versus CD3/CD28; #, P 0.001 versus VIP-treated samples.

Article Snippet: Protein A/G agarose and antibodies against CD3 chain, cyclin E, cyclin A, cdk2, cyclin D2, cyclin D3, cdk4, cdk6, Rap1, p27kip1, phospho-ERK1/2, p38, phospho-p38, c-Jun, and phospho-c-Jun were from Santa Cruz Biotechnology.

Techniques: Activity Assay, RIA Assay, Cell Cycle Assay, Expressing, Membrane, Phospho-proteomics, Activation Assay, Blocking 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: Neuropathology and Applied Neurobiology

Article Title: RAPGEF2 mediates oligomeric Aβ‐induced synaptic loss and cognitive dysfunction in the 3xTg‐AD mouse model of Alzheimer’s disease

doi: 10.1111/nan.12686

Figure Lengend Snippet: Knockdown of RAPGEF2 rescues fear memory deficits in 4‐month‐old 3xTg‐AD mice. A, Time course of stereotaxic delivery of lentiviral particles and behavioural tests. B, Representative image of GFP expression in the hippocampal CA1 area. Lentiviral particles simultaneously expressing shRAPGEF2 and GFP were bilaterally injected into the hippocampal CA1 area. Hoechst dye was used to stain neuronal nuclei (blue). Scale, 10 μm. Right, higher magnification image enclosed in rectangles at the left. A dotted line indicates the CA1 (mo: molecular layer in dentate gyrus). 53% ± 0.9% area of CA1 region was GFP‐positive (virus transduced) ( n = 3). Scale, 200 μm. C, Virus‐mediated knockdown extent of RAPGEF2 in vivo. Lentiviral particles expressing either shRAPGEF2 or a control empty vector (pll3.7) were transduced into hippocampal CA1 area in 2 months of wild‐type (WT) and 3xTg‐AD (TG) mice. After 2 months, hippocampal lysates were used for western blot analysis for RAPGEF2. APP‐FL (full length APP) were used as the marker of TG mice. D, Quantification of RAPGEF2 levels normalised to β‐Tubulin. pll3.7 in WT, n = 6 (5 males, 1 female); shRAPGEF2 in WT, n = 8 (7 males, 1 female); pll3.7 in TG, n = 4 (3 males, 1 female); shRAPGEF2 in TG, n = 8 (7 males, 1 female). E, The contextual fear memory test in 4‐month‐old wild‐type (WT) and 3xTg‐AD (TG) mice ( n = 6, males). F, Control (pll3.7) or shRAPGEF2‐expressing viral particles were injected into the hippocampal CA1 area in 2‐month‐old WT and 3xTg‐AD mice. After 2 months, the contextual fear memory test was performed (pll3.7 in WT, n = 15 males; shRAPGEF2 in WT, N = 8 males; pll3.7 in 3xTg‐AD mice, n = 22 males; shRAPGEF2 in 3xTg‐AD, n = 17 males). All data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01; **** p < 0.0001; two‐tailed unpaired Student's t ‐test (E) and Two‐way ANOVA, Tukey's multiple‐comparison test (D and F)

Article Snippet: Antibodies against phospho‐p38 (#4511), p38 (#9212), phospho‐JNK (#9255), JNK (#9252), Rap1 (#2399) and β‐tubulin (#2146) were purchased from Cell Signaling Technology.

Techniques: Knockdown, Expressing, Injection, Staining, Virus, In Vivo, Control, Plasmid Preparation, Western Blot, Marker, Two Tailed Test, Comparison