rap1a c 17  (Santa Cruz Biotechnology)

Journal: Genome Biology

Article Title: Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

doi: 10.1186/gb-2009-10-9-r93

Figure Lengend Snippet: N-BP-induced accumulation of unprenylated Rap1A and increase of DBF4, but not of TBCB, can be reversed by GGPP. Western blot (WB) analysis of MCF-7 cells treated with 10 -4 M ALE alone or in combination with 25 μM GGPP. The same volume of absolute ethanol was used as control vehicle of GGPP (Ctrl). Actin was used to show equal loading of the lanes.

Article Snippet: Blots were incubated with the following polyclonal antibodies: rabbit anti-Dbf4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-actin (Sigma), goat anti-Rap1A (C-17 - epitope mapping at the C-terminus of Rap 1A of human origin), and rabbit anti-Rap1 (121 - epitope mapping near the C-terminus of Rap 1 of human origin) (Santa Cruz Biotechnology), and anti-TBCB, a generous gift of JC Zabala, Universidad de Cantabria, Santander, Spain.

Techniques: Western Blot

Journal: The Journal of Biological Chemistry

Article Title: G Protein ?? Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.M109.069948

Figure Lengend Snippet: Rap1a-GTP and Gβγ promote the translocation of Radil at cell-matrix contacts in HT1080 cells. A, shown is localization of GFP (a and b), Venus-Radil (c and d), or Venus-Radil co-expressed with wild-type HA-Rap1 (e–g), HA-Rap1aQ63E (h–j), HA-Rap1G12V (k–m), or FLAG-Gβ2/Gγ1 (n–p). HA- and FLAG-tagged Rap1a or Gβ2 + Gγ (untagged) were transiently transfected in HT1080 cells stably expressing Venus-Radil as indicated. Cells were fixed followed by immunodetection with α-HA (e–m) or α-FLAG (n–p) monoclonal antibody followed by secondary detection with goat-anti-mouse conjugated to Alexa 594 antibody. Cells were visualized using a Zeiss LSM 510 confocal microscope under 63× oil immersion objective. White arrows depict plasma-membrane co-localization. White boxes indicate magnified sections cropped in Adobe Photoshop CS3. Images shown are representative of 10–15 cells analyzed in three independent experiments. Bars, 50 μm. B, membrane fractions show enrichment of Venus-Radil when Rap1aQ63E and Gβγ are expressed. HT1080 cells stably expressing Venus-Radil were transiently transfected to express the indicated proteins. Cells were lysed in hypotonic buffer and subjected to subcellular fractionation as described under “Experimental Procedures.” Membrane fractions and the inputs were analyzed by Western blot (WB) to determine the amount of Venus-Radil present in the membrane pool when HA-Rap1aQ63E or FLAG-Gβ2 was co-expressed. The blot for the membrane fractions was stripped and re-probed using α-Na-K-ATPase antibody to provide for an internal loading control.

Article Snippet: Antibodies were purchased from the following vendors: α-HA.11 (Covance), α-FLAG (Sigma), α-Gβ (T20) (Santa Cruz Biotechnology), α-Rap1a (C-17) (Santa Cruz Biotechnology), α-Erk1/2 (Cell Signaling), α-CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), α-GFP (Covance and the University of Alberta), α-tubulin (Sigma), and α-Na-K-ATPase (Affinity Bioreagents).

Techniques: Translocation Assay, Transfection, Stable Transfection, Expressing, Immunodetection, Microscopy, Fractionation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: G Protein ?? Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.M109.069948

Figure Lengend Snippet: Identification of Radil as a novel interactor of Gβγ subunits of heterotrimeric G proteins. A, shown is the protein-protein interaction network of Gβ2, Gγ2, and Radil. Single-headed arrows represent interactions found in Gβ2 (red), Gγ2 (blue), and Radil (purple) pulldown experiments, color coded according to the color of the bait. Dark double-sided arrows represent proteins reciprocally identified using the other as bait. Gβ2 (n = 3), Gγ2 (n = 2), and Radil pulldown assays (n = 2) were performed in HEK293T and HT1080 cells. Analysis of the tandem affinity-purified Gβ2 or Gγ2 protein complexes using mass spectrometry reveals several known Gβγ interactors. The small GTP-binding protein Rap1a and the newly characterized protein Radil were also identified in the Gβ2 complexes. The reciprocal analysis of Radil protein complexes confirmed Radil as a Gβγ-associated protein and also revealed that it binds small G proteins of the Ras family. B, streptavidin affinity purification (AP) of Strep-HA-Radil (left panel) and Strep-HA-Gγ2 (right panel) and immunodetection of co-purified endogenous Gβ, Rap1a, or Radil as indicated validates the mass spectrometry results. WB, Western blot. C, left panel, HEK293T cells were transiently transfected with expression vectors coding for FLAG-GFP, FLAG-Radil, or the closely related FLAG-AF6, and proteins were immunoprecipitated (IP)using α-FLAG M2-conjugated agarose beads followed by a Western blot with α-FLAG (top panel) or α-Gβ (bottom panel) antibodies (n = 3). Gβ co-immunoprecipitates with FLAG-Radil but not with FLAG-GFP or FLAG-AF6. Right panel, a schematic representation of Radil and AF6 proteins shows the similarity between the two proteins containing RA, DIL, and PDZ domains.

Article Snippet: Antibodies were purchased from the following vendors: α-HA.11 (Covance), α-FLAG (Sigma), α-Gβ (T20) (Santa Cruz Biotechnology), α-Rap1a (C-17) (Santa Cruz Biotechnology), α-Erk1/2 (Cell Signaling), α-CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), α-GFP (Covance and the University of Alberta), α-tubulin (Sigma), and α-Na-K-ATPase (Affinity Bioreagents).

Techniques: Affinity Purification, Mass Spectrometry, Binding Assay, Immunodetection, Purification, Western Blot, Transfection, Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: G Protein ?? Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.M109.069948

Figure Lengend Snippet: Interaction between Gβγ, Rap1a, and Radil. A, Radil physically connects Gβγ and Rap1a. HEK293T cells were transfected with different plasmid combinations coding for Strep-HA-Gβ2, Gγ2, HA-Rap1a, and Venus-Radil. Strep-HA-Gβ2-containing complexes were affinity-purified with streptavidin-Sepharose beads, and the association with Rap1a and Radil was monitored by Western blot (WB) with α-HA and α-GFP antibodies, respectively. The efficiency of Gβ2 purification and expression was also followed by Western blot using HA antibodies. A fraction of the lysates for each samples was probed with α-HA or α-GFP antibodies to assess protein expression. The association of Rap1a with Gβ2 was enhanced when Radil was overexpressed (compare lanes 2 and 3) (n = 3). *, nonspecific band. B, formation of the Gβγ·Rap1a·Radil complex requires active Rap1a. HEK293T cells were transfected with expression plasmids for Strep-HA-Radil and HA-Rap1a only (lane 2) or together with a vector coding for FLAG-Rap1GAP (lane 3). Strep-HA-Radil was purified using streptavidin-Sepharose beads, and its association with Rap1 and Gβ was monitored by a Western blot using α-HA and α-Gβ antibodies, respectively. The expression of Rap1GAP was followed using α-FLAG antibodies. Although HA-Rap1a and Gβ bound to Strep-HA-Radil (lane 2), these interactions were compromised in the presence of overexpressed FLAG-Rap1GAP (lane 3) (n = 4). C, expression of a constitutively active Rap1a mutant (Rap1aQ63E) promotes the interaction of Gβ with Radil. Strep-HA-Radil expressing stable cells were transfected or not with HA-Rap1aQ63E. 48 h after transfection, Radil was affinity-purified with streptavidin-Sepharose beads for 1.5 h, and the eluates were analyzed by Western blot using the indicated antibodies. Untransfected HEK293T cells were used as the negative control for streptavidin purification. AP, affinity purification. Mr, molecular weights. K, ×1000.

Article Snippet: Antibodies were purchased from the following vendors: α-HA.11 (Covance), α-FLAG (Sigma), α-Gβ (T20) (Santa Cruz Biotechnology), α-Rap1a (C-17) (Santa Cruz Biotechnology), α-Erk1/2 (Cell Signaling), α-CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), α-GFP (Covance and the University of Alberta), α-tubulin (Sigma), and α-Na-K-ATPase (Affinity Bioreagents).

Techniques: Transfection, Plasmid Preparation, Affinity Purification, Western Blot, Purification, Expressing, Mutagenesis, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: G Protein ?? Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.M109.069948

Figure Lengend Snippet: fMLP promotes Rap1-dependent HT1080 cell adhesion, Rap1 activation, and Radil translocation to the plasma membrane. A, HT1080 cell adhesion on a fibronectin matrix is shown with or without fMLP (100 nm) stimulation in the presence or absence of Rap1GAP. Rap1aQ63E (used as a positive control) shows the increase in cell adhesion (n = 3). B, shown is the time course of Rap1 activation by fMLP (100 nm) in HT1080 cells. The top panel shows GTP-bound Rap1a, purified using GST-RalGDS-RBD pulldown assays. The bottom panel shows total Rap1 in a fraction of whole cell lysates from each sample. Western blotting (WB) was done using α-Rap1a antibodies. C, HT1080 cells stably expressing Venus-Radil were serum-starved for 24 h and stimulated or not with fMLP (1 μm) for 0 min (a), 2 min (b), 3 min (c) at 37 °C. Arrows highlight regions of Venus-Radil enrichment at the plasma membrane. Images were captured by a blinded observer using 100× oil-immersion lens. Images shown are representative of two independent experiments. Bars, 20 μm.

Article Snippet: Antibodies were purchased from the following vendors: α-HA.11 (Covance), α-FLAG (Sigma), α-Gβ (T20) (Santa Cruz Biotechnology), α-Rap1a (C-17) (Santa Cruz Biotechnology), α-Erk1/2 (Cell Signaling), α-CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), α-GFP (Covance and the University of Alberta), α-tubulin (Sigma), and α-Na-K-ATPase (Affinity Bioreagents).

Techniques: Activation Assay, Translocation Assay, Positive Control, Purification, Western Blot, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: G Protein ?? Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

doi: 10.1074/jbc.M109.069948

Figure Lengend Snippet: Radil and Gβγ promote adhesion of HT1080 cells on fibronectin matrix in a Rap1a dependent manner. A, shown are representative pictures of HT1080 cell adhesion assays in response to overexpression of different proteins as indicated. FLAG-GFP cDNA was transfected as control. Top panels show the remaining adherent cells after washes. The bottom panels demonstrate the total cells input for each conditions. B, shown is a quantified representation of the adhesion assay as -fold stimulation compared with control. C, shown are representative pictures comparing the ability of ΔRA and ΔPDZ Radil mutants to promote HT1080 cells adhesion. D, shown is a quantified representation of C. The inset shows expression of the different Radil proteins in one representative experiment. Each experiment was done in triplicate, and several pictures from random fields were taken from each well of 96-well plate. Cells were counted (using an automated cell counting macro created in ImageJ software) from each field and expressed as -fold of control from each experiment. The results from three independent experiments were pooled for the quantification. Each dot on the graph represents cell counts from a single field. Error bars, ±S.E. Statistics are one-way ANOVA followed by Tukey's multiple comparison test; p < 0.05. Asterisks indicate statistical significance compared with control.

Article Snippet: Antibodies were purchased from the following vendors: α-HA.11 (Covance), α-FLAG (Sigma), α-Gβ (T20) (Santa Cruz Biotechnology), α-Rap1a (C-17) (Santa Cruz Biotechnology), α-Erk1/2 (Cell Signaling), α-CD29 monoclonal antibody (9EG7) (BD Transduction Laboratories), α-GFP (Covance and the University of Alberta), α-tubulin (Sigma), and α-Na-K-ATPase (Affinity Bioreagents).

Techniques: Over Expression, Transfection, Cell Adhesion Assay, Expressing, Cell Counting, Software

Journal: Molecular Cancer

Article Title: Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of Geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis

doi: 10.1186/1476-4598-9-23

Figure Lengend Snippet: RhoB is upregulationed by GGTI-298 (A) and in part mediates DR5 upregulation by GGTI-298 (B) . A , A549 cells were treated with 15 μM GGTI-298 for the given times and then subjected to preparation of whole-cell protein lysates and subsequent Western blotting. B , A549 cells were cultured in a 6-well plate and the next day transfected with the indicated siRNAs. After 36 h, the cells were treated with 15 μM GGTI-298 for 12 h and then subjected to preparation of whole-cell protein lysates for Western blotting. Ctrl, control; LE, longer exposure.

Article Snippet: Goat polyclonal anti-Rap1A (C-17; sc-1842) and rabbit polyclonal anti-RhoB (119) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Cell Culture, Transfection, Control