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

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Phosphorylation of Rap1 serine 731 modulates telomere length regulation. ( A ) Schematic diagram of Rap1 illustrates its domain structure and putative phosphorylation sites. The BRCT, linker, DNA-binding (Myb1 and Myb2), transactivation (TA) and regulatory C-terminal (RCT) domains are indicated. ( B ) Telomere analysis of rap1 mutants in YPH499 background. Genomic DNA from each colony was digested with KpnI, separated in a 1% agarose gel, transferred to a nylon membrane and hybridized with a TG 1–3 probe. The maximum integrity of the autoradiographic signal was determined by ImageQuant software and is indicated as a white line ( n = 2). ( C ) Deficiency of Rap1 S731 phosphorylation does not attenuate telomere position effect and mating type locus silencing. Mutation of RAP1 S731 was introduced into the URA3 reporter strains, UCC3505 (telomeric silencing), UCC3515 ( HML silencing) and UCC4564 ( HMR silencing). 10-fold serial dilution of cells was spotted onto YPAD, SC Ura − and SC 5-FOA plates. Inability to grow on 5-FOA plates indicates a loss of silencing effect. The isogenic rap1-ΔC and sir3 mutants were used as controls ( n = 2).

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques: Binding Assay, Agarose Gel Electrophoresis, Software, Mutagenesis, Serial Dilution

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Tel1/Mec1-mediated Rap1 phosphorylation on S731 is associated with telomere length variation. ( A ) DNA damage increases Rap1-S731 phosphorylation. Log phase cells were grown at 30°C in YPAD containing 50 mU/ml bleomycin or 0.05% Methyl methanesulfonate (MMS) for 3 h. Endogenous Rap1-Myc 13 proteins were immunoprecipitated and analyzed by western blotting. The Rap1 pS731 phospho-specific antibody detected the Rap1-S731 phosphorylation upon bleomycin and MMS treatment in WT. The levels of signal compared with that of the WT were shown below and expressed as the mean ± s.d. ( n = 3). The total Rap1 protein level was detected by the anti-Myc antibodies. ( B ) Telomere shortening increases Rap1-S731 phosphorylation. Rap1 S731 phosphorylation was examined for the individual colony from dissected tlc1 spore, which had senesced for about 75 generations (population doubling, PD75) and the yku80 spores. Endogenous Rap1-Myc 13 proteins were immunoprecipitated and analyzed by western blotting as in (A). The levels of signal compared with that of the WT were shown below and expressed as the mean ± s.d. ( n = 3). ( C ) Telomere lengthening decreases Rap1 S731 phosphorylation. The pif1-m2 and cdc13-S314A mutants showed the declined level of Rap1-S731 phosphorylation compared with that of the WT. Endogenous Rap1-Myc 13 proteins were immunoprecipitated and analyzed by western blotting as in (A). The levels of signal compared with that of the WT were shown below and expressed as the mean ± s.d. ( n = 3). ( D ) Rap1 S731 phosphorylation in vivo is Tel1- and Mec1-dependent. The Rap1 S731 phosphorylation levels were decreased in tel1 mec1 double mutants. Endogenous Rap1-Myc 13 proteins were immunoprecipitated and analyzed by western blotting as in (A). The levels of signal compared with that of the WT were shown below and expressed as the mean ± s.d. ( n = 3). ( E ) Tel1 and Mec1 phosphorylate Rap1 S731 in vitro . Left panel, in vitro Tel1 kinase assay was conducted using immunoprecipitants of HA-tagged Tel1 or kinase-dead mutant on recombinant GST-Rap1 (716–746) or GST-Rap1-S731A (716–746) substrates. Samples were loaded onto the 12% SDS-PAGE, and the phosphorylated proteins were detected by autoradiography (shown at the top, n = 3). The same gel was subsequently stained with Coomassie blue to confirm that all proteins were equally loaded (shown at the middle). The Tel1 kinases were resolved by 5% SDS-PAGE and western blotted with anti-HA antibodies (shown at the bottom, n = 3). Right panel, in vitro Mec1 kinase assay was performed as the left panel using Myc 18 -tagged Mec1 or kinase-dead mutants as kinases ( n = 3). The Mec1 kinases were resolved by 5% SDS-PAGE and western blotted with anti-Myc antibodies (shown at the bottom, n = 3). The levels of signal compared with that of the WT were shown below and expressed as the mean ± s.d.

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques: Immunoprecipitation, Western Blot, In Vivo, In Vitro, Kinase Assay, Mutagenesis, Recombinant, SDS Page, Autoradiography, Staining

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Telomere length variations of rap1-S731 mutants in the rif1, rif2 and rif1 rif2 backgrounds. ( A ) Telomere analysis of WT, rap1-S731A and rap1-S731D mutations in the rif1 background ( n = 2). ( B ) Telomere analysis of WT, rap1-S731A and rap1-S731D mutations in the rif2 background ( n = 2). ( C ) Telomere analysis of WT, rap1-S731A and rap1-S731D mutations in the rif1 rif2 background. The genomic DNA was processed and probed with the same approach as described in Figure ( n = 2).

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques:

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Rap1 S731A mutation reduces its interaction with Rif1, but not Rif2. ( A ) Yeast two-hybrid assay indicated that rap1-S731A mutation significantly reduces, whereas rap1-S731D increases, the Rap1–Rif1 interaction. The Y axis shows the relative folds of β-galactosidase activity. Error bars indicate the s.d. ( n = 4, * P -values < 0.05, ** P -values < 0.001, Student's t -test, two-tailed). ( B ) Yeast two-hybrid assay indicated that WT and rap1-S mutants display similar Rap1-Rif2 interaction ( n = 4, NS, non-significant, Student's t -test, two-tailed,). Bars, s.d. ( C ) Left panel, the aliquot of GST and GST-Rif1 (1709–1916) fusion proteins was resolved on SDS-PAGE and stained with Coomassie blue. Top-right panel, GST pulldown assay indicated that GST-Rif1 (1709–1916) significantly reduce its interaction with Rap1-S731A, whereas increase that with Rap1-S731D. Lower panel, the quantitative data of GST-Rif1 pulldown ( n = 4, * P -values < 0.05, ** P -values < 0.001, Student's t -test, two-tailed). Bars, s.d. ( D ) Left panel, the aliquot of GST and GST-Rif2 (1–395) fusion proteins was resolved on SDS-PAGE and stained with Coomassie blue. Top-right panel, GST pulldown assay demonstrated that the level of GST-Rif2 (1–395) interacting with Rap1 is not significantly different between WT and Rap1-S731A or Rap1-S731D, respectively. Lower panel, the quantitative data of GST-Rif2 pulldown ( n = 4, NS, non-significant, Student's t -test, two-tailed). Bars, s.d.

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques: Mutagenesis, Y2H Assay, Activity Assay, Two Tailed Test, SDS Page, Staining, GST Pulldown Assay

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Rif1, but not Rif2, occupancy at telomeres is reduced in rap1-S731A cells. ( A and B ). ChIP assay demonstrated that Rif1, but not Rif2, occupancy is lower at rap1-S731A versus WT telomeres. (A). The telomere binding level of Rif1 is significantly reduced in rap1-S731A cells ( n = 4, ** P -values < 0.001, Student's t -test, two-tailed). Strains expressing Myc-tagged proteins or untagged strain were immunoprecipitated with anti-Myc or anti-normal mouse IgG antibodies. Eluted DNA was analyzed by quantitative PCR to measure the occupancy of binding proteins on VI-R and XV-L telomeres. The data were presented as fold enrichment of telomeric sequence over the non-telomeric ARO1 sequence in the same samples. Bars, s.d. (B). The telomere binding level of Rif2 is indistinguishable between WT and rap1-S731A cells ( n = 4, NS, non-significant, Student's t -test, two-tailed). The experiment manipulations and data presentations were same as in (A). Bars, s.d. ( C ). ChIP assay showed that Rap1 content on VI-R and XV-L telomeres is not significantly different between WT and rap1-S731A cells ( n = 5, NS, non-significant, Student's t -test, two-tailed). Endogenous Rap1 was immunoprecipitated with anti-Rap1 antibodies. The data were presented as in (A). Bars, s.d. ( D ). Schematic model of Tel1 promoted Rap1–Rif1 interaction. Unphosphorylated Rap1-S731A causes diminished Rif1 binding to telomeres.

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques: Binding Assay, Two Tailed Test, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

Journal: Nucleic Acids Research

Article Title: Telomere shortening triggers a feedback loop to enhance end protection

doi: 10.1093/nar/gkx503

Figure Lengend Snippet: Tel1 orchestrates the telomere capping and telomerase recruitment pathways in telomere-shortened cells. The schematic model describes how telomere shortening enhances Rap1 phosphorylation and triggers a feedback loop to promote telomere end protection.

Article Snippet: Affinity-purified rabbit anti-Rap1 pS731 phospho-specific antibodies, in-house customized via GeneTex, were raised against CEVISGDYEPpSQAEK phosphopeptides to detect phosphorylation of Rap1 S731.

Techniques: