anti-rap2 rabbit polyclonal antibody  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

doi: 10.1371/journal.pone.0069289

Figure Lengend Snippet: HEK-293 cells were transfected either with RasGEF1B specific shRNA expressing vector (Sh-D, ), scrambled shRNA expressing vector, or empty vector (negative control), 48 hrs later cells were lysed as described at Materials and Methods, they were cleared by centrifugation, and active Rap was precipitated with a glutathione S -transferase fusion protein of the Ras-binding domain of RalGDS precoupled to glutathione-Sepharose beads. Rap2 activation assay were carried-out by using anti-Rap2 monoclonal Abs. Results confirmed that shRNA mediated down-regulation of RasGEF1B expression effectively block generation of Rap2 . GTP, although total cellular Rap2 was not affected. Calnexin expression was monitored as loading control.

Article Snippet: Sources of antibodies: Plk1 a gift from Nurhan Özlü, Koç University, Istanbul; monoclonal (ab17057), or polyclonal (ab109777) Abs were purchased from Abcam; anti-RasGEF1B-C-ter epitope Ab and anti-Ccdc124-N-ter epitope Ab were custom produced by Cambridge Research Biochemicals-UK (see above), Ccdc124 middle-epitope specific (A-301-835A) or C-ter epitope specific (A-301-834A) Abs were from BETHYL Laboratories; anti-γ-tubulin were either from Abcam (ab11316), or from BioLegend (rabbit polyclonal cat.620902); anti-α-tubulin were from Santa Cruz (SC-5286); rabbit anti-flag-Ab was from Sigma (F-7425); rabbit anti-Rap2 polyclonal was purchased from Genetex (GTX108831); mouse anti-Rap2 monoclonal was from BD Biosciences (610215), DNA dye (DAPI) was from Invitrogene (P36931); and Alexa Fluor 488, or 568 labeled anti-rabbit, and anti-mouse secondary antibodies were purchased from Invitrogene.

Techniques: Transfection, shRNA, Expressing, Plasmid Preparation, Negative Control, Centrifugation, Binding Assay, Activation Assay, Blocking Assay

Journal: PLoS ONE

Article Title: Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

doi: 10.1371/journal.pone.0069289

Figure Lengend Snippet: Subcellular localizations of endogenous Rap2 and Ccdc124 or RasGEF1B proteins were studied in HeLa cells by immunofluorescence methods. ( A ) At anaphase, Rap2 was clearly localized at the midzone, while Ccdc124 concentration at the same localization was less pronounced. However, at telophase, both proteins were concentrated at the puncta characterizing the midbody, Rap2 rather surrounding Ccdc124. During cytokinetic abscission, a clear colocalization of both Rap2 and Ccdc124 were observed at the midbody. ( B ) Similar to panel (A), Rap2 translocation to the midzone has started during anaphase. Two representative images of anaphase cells were shown in the corresponding panel. Both Rap2 and RasGEF1B proteins colocalized at the midbody during cytokinetic abscission. Arrowheads either indicate subcellular localization of Rap2 at anaphase and telophase, or they indicate the colocalization of Rap2 with RasGEF1B/Ccdc124 at the midbody during cytokinesis. ( C ) HeLa cells were transfected with shRNA vectors (Sh1, Sh2, Sh3) targeting Rap2, then cell lysates were collected at 48 hrs post-transfection, and immunoblotted with anti-Rap2 Ab. Scrambled control transfection was indicated (Scr). Calnexin expression was monitored as loading control. In parallel experiments, similarly treated cells were immunostained with anti-Rap2 and anti-α-tubulin antibodies, followed by scorings for multinucleation (n = 5± SD), as reported on the graph above the immunoblot. ( D ) Representative micrographs of midbody stage cells depleted in endogenous Rap2 (C). Bars represent 10 µm.

Article Snippet: Sources of antibodies: Plk1 a gift from Nurhan Özlü, Koç University, Istanbul; monoclonal (ab17057), or polyclonal (ab109777) Abs were purchased from Abcam; anti-RasGEF1B-C-ter epitope Ab and anti-Ccdc124-N-ter epitope Ab were custom produced by Cambridge Research Biochemicals-UK (see above), Ccdc124 middle-epitope specific (A-301-835A) or C-ter epitope specific (A-301-834A) Abs were from BETHYL Laboratories; anti-γ-tubulin were either from Abcam (ab11316), or from BioLegend (rabbit polyclonal cat.620902); anti-α-tubulin were from Santa Cruz (SC-5286); rabbit anti-flag-Ab was from Sigma (F-7425); rabbit anti-Rap2 polyclonal was purchased from Genetex (GTX108831); mouse anti-Rap2 monoclonal was from BD Biosciences (610215), DNA dye (DAPI) was from Invitrogene (P36931); and Alexa Fluor 488, or 568 labeled anti-rabbit, and anti-mouse secondary antibodies were purchased from Invitrogene.

Techniques: Immunofluorescence, Concentration Assay, Translocation Assay, Transfection, shRNA, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

doi: 10.1371/journal.pone.0069289

Figure Lengend Snippet: (A) HeLa cells were arrested at G2/M phase by sequential double thymidine and nocodazole treatments as described in the legend of , and they were classified according to phases of mitosis, and cytokinesis. Samples of cells were then stained with anti-Rap2 antibody, and with DAPI to visualize DNA. At anaphase Rap2 was detected at the midzone with staining characteristics reminiscent of endosomes, and at telophase/cytokinesis Rap2 was observed as puncta at the middle of the intercellular bridge, a position typically occupied by midbody associated factors. ( B ) Following synchronization of cells as above, 80 mins. after nocodazole was washed-off samples were taken with four consecutive intervals of 10 minutes (I, II, III, and IV), the last one (IV) corresponding to ∼120 minutes after the drug was removed, and dynamic positioning of Rap2 at the intercellular bridge in respect to α-tubulin was monitored. A time-dependent relocalization of Rap2 from peripheral flanking regions to the midbody was detected. Intercellular bridge localizations of Rap2 were concluded with observations from a sample of ∼50 cells in which over 75% showed similar positioning patterns. Two sets of representative micrographs were displayed. Bars represent 10 µm.

Article Snippet: Sources of antibodies: Plk1 a gift from Nurhan Özlü, Koç University, Istanbul; monoclonal (ab17057), or polyclonal (ab109777) Abs were purchased from Abcam; anti-RasGEF1B-C-ter epitope Ab and anti-Ccdc124-N-ter epitope Ab were custom produced by Cambridge Research Biochemicals-UK (see above), Ccdc124 middle-epitope specific (A-301-835A) or C-ter epitope specific (A-301-834A) Abs were from BETHYL Laboratories; anti-γ-tubulin were either from Abcam (ab11316), or from BioLegend (rabbit polyclonal cat.620902); anti-α-tubulin were from Santa Cruz (SC-5286); rabbit anti-flag-Ab was from Sigma (F-7425); rabbit anti-Rap2 polyclonal was purchased from Genetex (GTX108831); mouse anti-Rap2 monoclonal was from BD Biosciences (610215), DNA dye (DAPI) was from Invitrogene (P36931); and Alexa Fluor 488, or 568 labeled anti-rabbit, and anti-mouse secondary antibodies were purchased from Invitrogene.

Techniques: Staining

Journal: PLoS ONE

Article Title: Coiled-Coil Domain Containing Protein 124 Is a Novel Centrosome and Midbody Protein That Interacts with the Ras-Guanine Nucleotide Exchange Factor 1B and Is Involved in Cytokinesis

doi: 10.1371/journal.pone.0069289

Figure Lengend Snippet: (A) HeLa cells were transfected with the vector containing GFP-labeled Rap Binding Domain of RalGDS [GFP-RBD(RalGDS)] which interacts only with the GTP-bound active form of Rap2, and cells were monitored at interphase and at cytokinetic abscission following immunostainings involving anti-Rap2 monoclonal Abs and Alexa568-red labeled anti-mouse secondary Abs. Colocalization of GFP-RBD(RalGDS) with Rap2 indicated that at the midbody Rap2 is in its active (Rap2 . GTP) form. ( B ) HeLa cells were cotransfected with GFP-RBD(RalGDS) either together with HA-Rap2-WT, or with HA-Rap2-S17N (inactive dominant negative form) and their localizations were monitored with anti-HA-epitope Abs. Positioning of GFP-RBD(RalGDS) were assessed by monitoring GFP-signal observed at the midbody. Localization of the GFP-RBD(RalGDS) depended on the presence of active Rap2 in cells. As in (A), at least 50 cells were monitored in each experiment, and representative pictures display Rap2 and GFP-RBD(RalGDS) localizations observed at least ∼90% of cells cotransfected with indicated vectors. Bars represent 10 µm.

Article Snippet: Sources of antibodies: Plk1 a gift from Nurhan Özlü, Koç University, Istanbul; monoclonal (ab17057), or polyclonal (ab109777) Abs were purchased from Abcam; anti-RasGEF1B-C-ter epitope Ab and anti-Ccdc124-N-ter epitope Ab were custom produced by Cambridge Research Biochemicals-UK (see above), Ccdc124 middle-epitope specific (A-301-835A) or C-ter epitope specific (A-301-834A) Abs were from BETHYL Laboratories; anti-γ-tubulin were either from Abcam (ab11316), or from BioLegend (rabbit polyclonal cat.620902); anti-α-tubulin were from Santa Cruz (SC-5286); rabbit anti-flag-Ab was from Sigma (F-7425); rabbit anti-Rap2 polyclonal was purchased from Genetex (GTX108831); mouse anti-Rap2 monoclonal was from BD Biosciences (610215), DNA dye (DAPI) was from Invitrogene (P36931); and Alexa Fluor 488, or 568 labeled anti-rabbit, and anti-mouse secondary antibodies were purchased from Invitrogene.

Techniques: Transfection, Plasmid Preparation, Labeling, Binding Assay, Dominant Negative Mutation