rap1 specific antibody  (Novus Biologicals)

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:

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