rabbit polyclonal anti-rap1a  (ABclonal Biotechnology)

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) Patient breast tumors from Stages I–IV of the disease express higher levels of Rap1A mRNA compared to non-diseased Stage 0 samples, as determined by quantitative real-time PCR. Data points shown are log-transformed values of relative mRNA expressions compared to average expression in Stage 0 samples. Levels of β-actin were used by the manufacturer to pre-normalize samples. One-way ANOVA statistical analysis was performed on the log-transformed relative expression values and a Dunnett’s multiple comparison post-hoc test was conducted. a, p<0.01, compared to Stage 0. (B) Immunofluorescence and confocal microscopy on formalin-fixed and paraffin-embedded human tissue samples using (B) a rabbit anti-Rap1A polyclonal antibody followed by Alexa-Fluor 488 secondary antibody (green) and (C) a goat anti-β-arrestin2 polyclonal antibody followed by Alexa-Fluor 555 secondary antibody. In order to distinguish cells of epithelial origin, staining for cytokeratin CAM 5.2 was performed using a mouse monoclonal anti-CAM 5.2 antibody (red). Hematoxylin and eosin staining of normal and tumor sections from each group were examined (representative red boxes). Images are representative of four independent experiments. Scale bar, 20 µm.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Real-time Polymerase Chain Reaction, Transformation Assay, Expressing, Comparison, Immunofluorescence, Confocal Microscopy, Staining

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) The aggressive breast cancer cell lines MDA-MB-231 and Hs578T express elevated levels of Rap1A protein relative to the non-invasive MCF-7 cells or the non-malignant MCF-10A mammary epithelial cells. Endogenous expression of Rap1A in each cell line was determined by Western blot analysis. A representative blot from five separate experiments is shown. In the graphs, bars represent relative expression to MCF-10A cells. Error bar, SEM *, p<0.05. (B) Knockdown of Rap1A protein expression in MDA-MB-231 cells stably expressing two separate Rap1A shRNA constructs was verified by Western blot analysis. Densitometric analysis shows significantly lower levels of Rap1A expression in Rap1A shRNA expressing cells. *, p<0.05. (C) Knock-down of endogenous Rap1A in serum-starved MDA-MB-231 cells inhibits LPA (10 µM) stimulated cell invasion as assessed using Transwell chamber (Matrigel) invasion assays. (D) MDA-MB-231 cells expressing Rap1A shRNA and suspendend in Matrigel for 5 days display reduced growth of invasive stellate structures relative to cells expressing scrambled control. Nuclei were stained using Hoechst 33258 and visualized by confocal microcopy (blue, last column of panel). Images are representative of five independent experiments, scale bar = 40 µm. Shape of the colonies was scored as either stellate or spheroidal and the number of stellate colonies is expressed as percentage of the total number of colonies. *, p<0.05 compared to formation of stellate structures by MDA-MB-231 cells expressing scrambled shRNA.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Expressing, Western Blot, Knockdown, Stable Transfection, shRNA, Construct, Control, Staining

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) Depletion of Rap1A expression significantly blocks scratch closure of MDA-MB-231 cells in response to LPA treatment. Cells were grown to confluence, serum starved for four hours and scratched with a sterile pipette tip. Serum-free medium containing 10 µM LPA was added to the cells which allowed to migrate into the scratch for 16 hours and visualized using an IX-81 Olympus microscope. Cell migration was quantitated using ImagePro software and bars represent the mean distance travelled (in µm) after 16 hours of culturing in LPA. Data from four experments.*, p<0.05, scale bar = 100 µm. (B) LPA (10 µM) stimulates lamellipodia formation at the leading edge of motile MDA-MB-231cells expressing scrambled shRNA (control, upper panels) in a scratch assay. The effect of depleting endogenous Rap1A from LPA-stimulated cells is shown (lower panel). Cells, grown to confluency were serum starved for 4h and scratched with a sterile pipette tip. Cells were then placed in media containing LPA and allowed to migrate into the scratch for 4 hours, fixed, permeabilized, immunostained and visualized using confocal microscopy. Alexa-Fluor 555 conjugated phalloidin (red) was used to stain F-actin. Loss of color (grayscale image) shows the lamellipodia at the leading edge (white arrowheads). Direction of cell migration shown by blue arrow. Scale bar, 20 µm. Quantitation of cells with lamellipodia was carried out as described under “ ” and graphed. Error bar, SEM *, p<0.05. Data from three independent experiments. (C) Migration of serum-starved, Rap1A-depleted MDA-MB-231 cells towards 10 µM LPA is significantly inhibited, as determined by Transwell chamber assays. *, p<0.05. Data from four experments. (D) Confocal micrographs showing the localization of LPA 1 in MDA-MB-231 cells stably expressing FLAG-LPA 1 subjected to a scratch assay, as described above. Immunostaining for FLAG (red) with a rabbit anti-FLAG antibody; nuclei were stained using Hoechst 33258. (E) LPA 1 interacts with endogenous Rap1A. Co-immunoprecipitation studies were performed using serum-starved (4h) cells stably expressing FLAG-LPA 1 and using a mouse anti-FLAG monoclonal antibody. Endogenous Rap1A was detected by a rabbit polyclonal anti-Rap1A antibody. A representative blot from three independent experiments is shown. Graph illustrates densitometric analysis of blots; data are expressed relative to unstimulated cells. Error bar, SEM *, p<0.05.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Expressing, Sterility, Transferring, Microscopy, Migration, Software, shRNA, Control, Wound Healing Assay, Confocal Microscopy, Staining, Quantitation Assay, Stable Transfection, Immunostaining, Immunoprecipitation

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) Depletion of β-arrestin2 in breast cancer cells blocks LPA-induced Rap1 activation. MDA-MB-231 cells stably expressing either scrambled shRNA or β-arrestin2-targeting shRNA were serum starved for 4 hours and stimulated with 10 µM LPA. Cells were lysed and activated Rap1 was pulled down. Knockdown of β-arrestin2 in MDA-MB-231 cells stably expressing shRNA against β-arrestin2 was verified by Western blot analysis. Representative blots from four independent experiments are shown. (B) The association of endogenous proteins, β-arrestin2, Rap1A and IQGAP1, in breast cancer cells. MDA-MB-231 cells were serum-starved for 4 hours and stimulated with LPA in serum-free media (10 µM). Isolated lysates were subjected to co-immunoprecipitation assays using a rabbit anti-Rap1A antibody or rabbit anti-IQGAP1 antibody and immunoblotted using a monoclonal anti-β-arrestin2 antibody. A representative blot from four independent experiments is shown. Graph illustrates densitometric analysis of blots; data are expressed relative to unstimulated cells. Error bar, SEM *, p<0.05. (C) Endogenous proteins, β-arrestin2, Rap1A and IQGAP1 association in Hs578T cells as described in (B). A representative blot from three independent experiments is shown.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Activation Assay, Stable Transfection, Expressing, shRNA, Knockdown, Western Blot, Isolation, Immunoprecipitation

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) Endogenous IQGAP1 binds Rap1A in MDA-MB-231 breast cancer cells. Serum-starved (4h) cells stably expressing YFP-Rap1A or GFP vector control were stimulated with LPA (10 µM) and lysates were subjected to immunoprecipitation assays with an anti-GFP monoclonal antibody and immunoblotted with a rabbit anti-IQGAP1 polyclonal antibody. (B) IQGAP1 does not associate with RalA in MDA-MB-231. Serum starved (4h) cells were treated with 10 µM LPA and co-immunoprecipitation studies done with an anti-GFP monoclonal antibody. Endogenous IQGAP1 was detected using a rabbit anti-IQGAP1 antibody. (C) LPA 1 interacts with endogenous IQGAP1. Co-immunoprecipitation studies were performed using serum-starved (4h) cells stably expressing FLAG-LPA 1 and using a mouse anti-FLAG monoclonal antibody. Endogenous IQGAP1 was detected by a rabbit polyclonal anti-IQGAP1 antibody. (D) Depletion of IQGAP1 blocks LPA-induced cell migration and invasion. Serum starved (4h) MDA-MB-231 cells expressing IQGAP1 siRNA were subjected to Transwell migration or Matrigel-invasion assays towards 10 µM LPA (in serum-free media) . Results represent data from three independent experiments. *, p<0.05. IQGAP1 expression in MDA-MB-231 cells stably expressing siRNA against IQGAP1 was verified by Western blot analysis. A representative blot from three independent experiments is shown.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Migration, Western Blot

Journal: PLoS ONE

Article Title: β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

doi: 10.1371/journal.pone.0056174

Figure Lengend Snippet: (A) MDA-MB-231 cells were serum starved for 4h and scratched with a sterile pipette tip. Cells were then placed in serum-free medium containing 10 µM LPA and allowed to migrate into the scratch. Cells were fixed, permeabilized, immunostained and visualized using confocal microscopy. LPA (10 µM) stimulates lamellipodia formation and the accumulation of IQGAP1 at the leading edge of motile MDA-MB-231cells (white arrowheads) expressing scrambled shRNA (control, upper panel) in a scratch assay. The effect of depleting endogenous β-arrestin2 from LPA-stimulated cells is shown (lower panel). Endogenous IQGAP1 immunostaining was performed with rabbit anti-IQGAP1 antibody followed by Alexa-Fluor 488 (green). Scale bar, 20 µm. (B) β-arrestin2 expression in MDA-MB-231 cells stably expressing shRNA against β-arrestin2 was verified by Western blot analysis. A representative blot from three independent experiments is shown. Graph showing blind quantification of cells containing IQGAP1 at the leading edge of migrating cells from (A) carried out after stimulation of cells with LPA for 4 hours, as described under “Experimental Procedures”. Error bar, SEM *, p<0.05. Data from three independent experiments. (C) Depletion of IQGAP1 blocks the endogenous association of Rap1A and β-arrestin2 in MDA-MB-231 cells. Co-immunoprecipitation studies were performed using a rabbit polyclonal anti-Rap1A antibody. Endogenous β-arrestin2 was detected with a monoclonal anti-β-arrestin2 antibody. A representative blot from three independent experiments.

Article Snippet: Serial sections were de-paraffinized, cleared, rehydrated through an ethanol series, and subjected to either hematoxylin and eosin staining or immunofluorescence (IF) using polyclonal anti-Rap1A rabbit antibody (1:50, Santa Cruz), polyclonal anti-β-arrestin2 goat antibody (1:50, C-18, Santa Cruz) and monoclonal anti-CAM5.2 mouse antibody (undiluted, BD Biosciences).

Techniques: Sterility, Transferring, Confocal Microscopy, Expressing, shRNA, Control, Wound Healing Assay, Immunostaining, Stable Transfection, Western Blot, Immunoprecipitation

Journal: Leukemia

Article Title: Blocking protein geranylgeranylation is essential for lovastatin-induced apoptosis of human acute myeloid leukemia cells.

doi: 10.1038/sj.leu.2402196

Figure Lengend Snippet: Figure 7 GGPP rescues lovastatin-induced inhibition of protein geranylgeranylation in AML-3 cells. Cells were exposed to solvent control (lane 1), 20 m lovastatin (lane 2), 20 m lovastatin + 10 m GGPP (lane 3), 20 m lovastatin + 10 m FPP (lane 4), 20 m GGTI- 298 (lane 5) and 20 m FTI-277 (lane 6) for 48 h. The cells were then harvested and cell lysates were subjected to SDS-polyacrylamide gel and immunoblotted for Rap1A and Rab5 using respective antibodies. Detection was performed by the ECL system (for the details, see Materials and methods).

Article Snippet: Western blotting was performed using a polyclonal rabbit anti-Rap1A antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a mouse monoclonal anti-Rab5 antibody (Transduction Laboratories, Lexington, KY, USA).

Techniques: Inhibition, Solvent, Control