Journal:

Article Title: A Longitudinal Study of Human Antibody Responses to Plasmodium falciparum Rhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission

doi:

Figure Lengend Snippet: Schematic representation showing mature P. falciparum RAP1 and rRAP1 proteins. P2 to P5 are proteins fused to hexahistidine tag (■); C1 and C2 are fused to GST (). , region of repeats related to the KSSSPS motif (between amino acid 123 and 164). ↑, cleavage site (between amino acid 190 and 191) of mature RAP1 to yield p65 fragment (35). private-char pc1, inhibitory monoclonal antibody epitope (L202 TPLEELYP210 [16]). , cysteine-rich region (between amino acid 353 and 616). Amino acid numbering is as in the work of Ridley et al. (37). This figure has been modified from the work of Fonjungo et al. (13).

Article Snippet: The procedure for determining specific anti-RAP1 IgM antibodies was similar to that described above for IgG except that horseradish peroxidase-conjugated rabbit anti-human IgM (Dako Ltd.) diluted 1:2,000 was used for detecting IgM antibodies.

Techniques: Modification

Journal:

Article Title: A Longitudinal Study of Human Antibody Responses to Plasmodium falciparum Rhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission

doi:

Figure Lengend Snippet: Antibody response to RAP1 in the Daraweesh cohort

Article Snippet: The procedure for determining specific anti-RAP1 IgM antibodies was similar to that described above for IgG except that horseradish peroxidase-conjugated rabbit anti-human IgM (Dako Ltd.) diluted 1:2,000 was used for detecting IgM antibodies.

Techniques: Infection

Journal:

Article Title: A Longitudinal Study of Human Antibody Responses to Plasmodium falciparum Rhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission

doi:

Figure Lengend Snippet: Different patterns of IgG antibody responses to RAP1 in natural P. falciparum infections. Each panel illustrates the time course of response(s) of one individual as detected by ELISA with six rRAP1 proteins. The x axes show the month and the year when each test sample was collected. Dates of samples collected during documented episodes of clinical malaria are underlined. PCR results, also along the x axes, indicate the absence (−) or presence (+) of P. falciparum in blood samples of each individual. nd, PCR was not done.

Article Snippet: The procedure for determining specific anti-RAP1 IgM antibodies was similar to that described above for IgG except that horseradish peroxidase-conjugated rabbit anti-human IgM (Dako Ltd.) diluted 1:2,000 was used for detecting IgM antibodies.

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: A Longitudinal Study of Human Antibody Responses to Plasmodium falciparum Rhoptry-Associated Protein 1 in a Region of Seasonal and Unstable Malaria Transmission

doi:

Figure Lengend Snippet: IgG responses to RAP1 are short-lived after recovery from P. falciparum infection. Reactivities of rRAP1 antigens with IgG in sera donated by five different individuals on the indicated dates before, during, and after drug-cured clinical malarial episodes (asterisks) are shown. Note a rapid decline of antibody levels in all cases. A cutoff value indicated by a horizontal line is that of P4; cutoffs for the other antigens were lower (see Materials and Methods).

Article Snippet: The procedure for determining specific anti-RAP1 IgM antibodies was similar to that described above for IgG except that horseradish peroxidase-conjugated rabbit anti-human IgM (Dako Ltd.) diluted 1:2,000 was used for detecting IgM antibodies.

Techniques: Infection

Journal: Science signaling

Article Title: Rapgef2 Connects GPCR-Mediated cAMP Signals to ERK Activation in Neuronal and Endocrine Cells

doi: 10.1126/scisignal.2003993

Figure Lengend Snippet: (A) Measurement of Rap1 activation in NS-1 cells pretreated with vehicle, 100 μM ddAd, 30 μM FTS-A, or 10 μM U0126 before being treated with vehicle or 100 nM PACAP-38 for 5 min. (B) Measurement of Rap1 activation in cells treated with either 100 μM 8-CPT-cAMP or 100 μM 8-CPT-2′-O-Me-cAMP (007) in the presence or absence of 30 μM H-89, 1 mM SQ22,536, or both. Western blotting data are representative of four experiments. Bar graph shows the mean ratios of the abundance of activated Rap1 (Rap1-GTP) to that of total Rap1 protein in each sample and are expressed relative to the amount of activated Rap1 in untreated control cells. #P < 0.05 (Bonferroni). (C) FTS-A inhibited activation of a reporter gene for Elk1 after treatment with 8-Br-cAMP (500 μM). (D) Inhibition of 8-Br-cAMP–dependent Elk1 induction in NS-1 cells by the B-Raf inhibitor PLX4720 and the MEK inhibitor U0126. Points represent means from three experiments, and error bars correspond to SEM. (E) Analysis of ERK activation. NS-1 cells were pretreated with vehicle, 30 μM FTS-A, 10 μM PLX4720, or 30 μM H-89 before being treated with vehicle or 100 nM PACAP-38 for 10 min, and the extent of ERK phosphorylation was determined by Western blotting analysis with antibodies against the indicated proteins. Western blots are representative of four experiments. Bar graph shows the ratio of the abundance of pERK to that of total ERK protein for each sample relative to untreated control cells (set at 100%). **P < 0.01 (Bonferroni), compared to untreated controls.

Article Snippet: Samples were then centrifuged through spin cups, washed three times with lysis buffer, dissolved in reducing sample buffer, vortexed, and heated to 95°C for 5 min. All samples were then resolved by 12% PAGE, gels were blotted onto nitrocellulose, and membranes were incubated with an antibody specific for Rap1 at a dilution of 1:1000 (Upstate Biotechnology).

Techniques: Activation Assay, Western Blot, Control, Inhibition, Phospho-proteomics

Journal: Science signaling

Article Title: Rapgef2 Connects GPCR-Mediated cAMP Signals to ERK Activation in Neuronal and Endocrine Cells

doi: 10.1126/scisignal.2003993

Figure Lengend Snippet: (A) Knockdown of Rapgef2 protein in NS-1 cells transduced with lentivirus expressing Rapgef2-specific shRNA. Lane 1: NS-1 cells expressing scrambled shRNA; lanes 2 to 4: stable NS-1 cell lines expressing Rapgef2-specific shRNA constructs. Cells in lane 4 contained <20% of the Rapgef2 protein in cells expressing scrambled shRNA and were propagated for further analyses. (B) Measurement of ERK phosphorylation in NS-1 cells expressing either scrambled shRNA or Rapgef2-specific shRNA. Cells were treated for 10 min with 100 nM PACAP-38, 25 μM forskolin, cholera toxin (CTX; 50 μg/ml), 100 μM 8-CPT-cAMP (8-CPT), NGF (100 ng/ml), or FGF (100 ng/ml). In cells expressing scrambled shRNA, all agents tested caused a statistically significant increase in ERK phosphorylation compared to that in untreated cells (Bonferroni, *P < 0.05, ***P < 0.001). In cells expressing Rapgef2-specific shRNA, only NGF and FGF caused statistically significant ERK phosphorylation (***P < 0.001). Lower panel shows a Western blot that is representative of four independent experiments. (C) Quantification of neurite outgrowth. NS-1 cells expressing either scrambled shRNA or Rapgef2-specific shRNA were treated for 48 hours with 100 nM PACAP-38, 25 μM forskolin, CTX (50 μg/ml), NGF (100 ng/ml), or FGF (100 ng/ml). In cells expressing scrambled shRNA, all agents tested caused significant neurite outgrowth relative to that of untreated control cells (Bonferroni, **P < 0.01, ***P < 0.001). Only NGF and FGF caused statistically significant neurite extension in cells stably expressing Rapgef2-specific shRNA. Bars represent means ± SEM from four fields per condition in three independent experiments. Representative photomicrographs are shown in fig. S6. (D) The association between Rap1 and B-Raf is Rapgef2-dependent. NS-1 cells expressing either scrambled shRNA or Rapgef2-specific shRNA were treated with 100 μM 8-CPT-cAMP or 100 μM 8-CPT-2′-O-Me-cAMP for 10 min. Protein content in cell lysates was normalized, and lysates were precleared and resolved by SDS-PAGE (polyacrylamide gel electrophoresis). Gels were blotted onto nitrocellulose and incubated with antibodies raised against B-Raf and Rap1. Lysates were also subjected to coimmunoprecipitation with a B-Raf–specific antibody coupled to agarose resins. The association between Rap1 and B-Raf in response to 8-CPT-cAMP was greater in extent in cells expressing scrambled shRNA than in cells expressing Rapgef2-specific shRNA. Data are representative of three experiments.

Article Snippet: Samples were then centrifuged through spin cups, washed three times with lysis buffer, dissolved in reducing sample buffer, vortexed, and heated to 95°C for 5 min. All samples were then resolved by 12% PAGE, gels were blotted onto nitrocellulose, and membranes were incubated with an antibody specific for Rap1 at a dilution of 1:1000 (Upstate Biotechnology).

Techniques: Knockdown, Transduction, Expressing, shRNA, Construct, Phospho-proteomics, Western Blot, Control, Stable Transfection, SDS Page, Polyacrylamide Gel Electrophoresis, Incubation

Journal: Science signaling

Article Title: Rapgef2 Connects GPCR-Mediated cAMP Signals to ERK Activation in Neuronal and Endocrine Cells

doi: 10.1126/scisignal.2003993

Figure Lengend Snippet: (A) Profile of Rapgef2 protein abundance in human and rodent cell lines. Lane 1: PC12-G; lane 2: NS-1; lane 3: BCC; lane 4: 293T rPAC1hop; lane 5: HEK 293FT; lane 6: HeLa; lane 7: 3T3 Swiss; lane 8: SH-SY5Y; lane 9: NG108-15; lane 10: NBFL; lane 11: FRTL-5; lane 12: B16-F10; lane 13: AtT20; lane 14: Chinese hamster ovary (CHO) K1. Western blotting data are representative of three independent experiments. (B) Western blotting analysis of Rapgef2 abundance in HEK 293T rPAC1hop cells (lane 1), HEK 293T rPAC1hop cells stably expressing hRapgef2 (lane 2), and nontransduced SH-SY5Y cells (lane 3). Western blotting data are representative of three independent experiments. (C and D) Measurement of ERK phosphorylation in (C) 293T rPAC1hop cells and (D) 293T rPAC1hop cells expressing hRapgef2 after treatment for 10 min with 100 nM PACAP-38, 30 μM isoproterenol, 25 μM forskolin, cholera toxin (CTX; 50 μg/ml), 0.5 mM 8-Br-cAMP, or 100 nM PMA. Bar graphs show the ratio of the abundance of pERK to that of total ERK protein for each sample relative to untreated control cells (set at 100%). *P < 0.05, **P < 0.01, ***P < 0.001; n = 4 experiments. (E) Analysis of Rap1 activation in 293T rPAC1hop cells expressing or not expressing the retroviral vector encoding hRapgef2. Cells were treated with vehicle, 100 μM 8-CPT-cAMP, or 100 μM 8-CPT-2′-O-Me-cAMP. *P < 0.05, **P < 0.01, compared to vehicle-treated cells. In cells expressing hRapgef2, 8-CPT-cAMP conferred a significantly greater activation of Rap1 (**P < 0.01) than was observed in nontransduced cells (#P < 0.05, Bonferroni). Data are from four to six independent experiments.

Article Snippet: Samples were then centrifuged through spin cups, washed three times with lysis buffer, dissolved in reducing sample buffer, vortexed, and heated to 95°C for 5 min. All samples were then resolved by 12% PAGE, gels were blotted onto nitrocellulose, and membranes were incubated with an antibody specific for Rap1 at a dilution of 1:1000 (Upstate Biotechnology).

Techniques: Quantitative Proteomics, Western Blot, Stable Transfection, Expressing, Phospho-proteomics, Control, Activation Assay, Retroviral, Plasmid Preparation

Journal: PLoS ONE

Article Title: Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo

doi: 10.1371/journal.pone.0073070

Figure Lengend Snippet: (A) In vitro , CECs have decreased transmigration across RPE monolayers when RPE are incubated with 8CPT-CAMP. Representative experiment of 2 independent trials. Data plotted as the average number of transmigrated CECs (± SEM) per 3 random fields, n = 6 Transwells per condition. * p<0.001 (B) Representative confocal images (maximum projections) of lectin-stained RPE/choroid flat mounts, 1 week post laser and intravitreal injection of PBS or 20.5 µM 8CPT-cAMP. (C) Quantification: intravitreal injection of 8CPT-cAMP induces a dose-dependent decrease in CNV volume in compared to PBS-injected control. *p<0.01, **p<0.001 (n = 13–21 lesions per condition) (D) Intravitreal injection of 8CPT-cAMP activates Rap1 in vivo . RPE/choroid was dissected and lysates were blotted with an antibody that detects active Rap1 (GTP-bound), or β-actin as loading control. Graph shows densitometry of active Rap1 normalized to β-actin (average ± SEM, n = 6). * p<0.0001.

Article Snippet: Supernatants were used for Western blotting using an antibody specific for active (GTP-bound) Rap1 (NewEast, PA), and β-actin (Santa Cruz, CA).

Techniques: In Vitro, Transmigration Assay, Incubation, Staining, Injection, Control, In Vivo

Journal: PLoS ONE

Article Title: Rap1 GTPase Activation and Barrier Enhancement in RPE Inhibits Choroidal Neovascularization In Vivo

doi: 10.1371/journal.pone.0073070

Figure Lengend Snippet: Laser treatment creates a breach in RPE and Bruch's membrane; RPE barrier integrity is compromised in cells adjacent to the lasered region. Inflammatory and wound healing events lead to activation of CECs from the choriocapillaris; CECs begin to migrate and transmigrate the lasered lesion as well as the adjacent RPE with compromised barrier integrity. In Type 2 CNV (shown), CECs proliferate and invade the subretinal space to form CNV. Type 1 (occult) CNV occurs when CECs remain sub-RPE (not shown in this model). 8CPT-cAMP injection post-laser inhibits CNV by promoting barrier integrity in the neighboring RPE, thereby reducing the lesion width through which CECs migrate. CEC junctional integrity may also be strengthened, contributing to the decreased CNV. Compared to WT, Rap1b −/− RPE cell junctions are more easily disrupted, allowing greater CEC transmigration and increased CNV. 8CPT-cAMP treatment activates both Rap1 isoforms, which is associated with increased junctional resealing and limits RPE monolayer disruption through which CEC migration occurs.

Article Snippet: Supernatants were used for Western blotting using an antibody specific for active (GTP-bound) Rap1 (NewEast, PA), and β-actin (Santa Cruz, CA).

Techniques: Membrane, Activation Assay, Injection, Transmigration Assay, Disruption, Migration

Journal: iScience

Article Title: Rap1 organizes lymphocyte front-back polarity via RhoA signaling and talin1

doi: 10.1016/j.isci.2023.107292

Figure Lengend Snippet:

Article Snippet: Antibodies specific for Rap1 and CDC42 were from BD Transduction Laboratories.

Techniques: Purification, Transduction, Recombinant, Cell Isolation, Western Blot, Mutagenesis, Software