rabbit polyclonal anti grb7  (Santa Cruz Biotechnology)

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A-C. A) Top: Domain topology of the Grb7 protein. The approximate locations of the Grb7 protein domains described in the text are identified by residue numbers located above the Grb7 schematic. The GM (Grbs and Mig) domain corresponds roughly to the homologous regions between the Grb7 and Mig 10 proteins. Bottom: Domain topology of the Filamin-a protein (dimer). The Filamin-a figure was adapted from Nakamura et al., 2011. B-C: β-galactosidase assays. B) The prey vector containing the IgFlna-16-19-AD (activation domain) was cotransformed in yeast with bait vectors containing the Grb7-RA-DBD (DNA binding domain), Grb7-PH-DBD, or Grb7-RA-PH-DBD, respectively (Figure shows the Grb7-RAPH-DBD experiment only). The p53/SV-40-T interaction serves as a positive control. C) β-galactosidase assay activity was determined by liquid assays using ONPG as substrate. The data represent averages with standard deviations from three independent experiments each done in triplicate.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Plasmid Preparation, Activation Assay, Binding Assay, Positive Control, Activity Assay

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A) GST binding assay: the IgFlna-16-19 domains bind with the Grb7-RA-PH domains. GST-IgFlna-16-19 and Grb7-RA-PH-6His were bacterially expressed and purified using glutathione (Glu-) sepharose beads and Ni-NTA agarose beads, respectively. Grb7-RA-PH-6His was incubated with GST-IgFlna-16-19 coupled glutathione beads and the eluted fraction was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained using Coomassie brilliant blue. Lanes: 1: protein standards, 2: purified GST-IgFlna-16-19, 3: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 4: purified Grb7-RA-PH-6His, 5: Glu-sepharose beads elution fraction of GST-IgFlna-16-19 and Grb7-RA-PH-6His mixture, 6: Glu-sepharose beads elution fraction of GST and Grb7-RA-PH-6His mixture, 7: Glu-sepharose beads elution fraction of Grb7-RAPH-6His alone. B) in vitro pull down of myc-Grb7-RA-PH by IgFlna-16-19. in vitro translated myc-tagged Grb7- RA-PH was incubated with purified glutathione coupled GST-IgFlna-16-19 (Lane 1) or GST alone (Lane 2). Lane 3 represents the elution fraction from glutathione coupled GST-IgFlna-16-19 only. Elution fractions were resolved by SDS-PAGE, and proteins were identified by Western blot analysis using mouse myc monoclonal antibody for Grb7-RA-PH detection and mouse GST monoclonal antibody for IgFlna-16-19 detection. C) In vivo coimmunoprecipitation of endogenous Grb7 and Filamin-a proteins. Left panels: coimmunoprecipitation with resin-coupled Grb7 antibody was performed on SKBR3 cell extracts endogenously expressing Grb7 and Filamin-a (Lanes 1–2, left upper panel). The whole cell lysate (WCL, lower left panel) and the resulting immunocomplexes (IP: Grb7) were immunoblotted with anti Grb7 (H-70), anti-Filamin-a (3 F-180), and anti-Actin (C-2) antibodies. A non-specific IgG control antibody immunoprecipitation (Lane 3, upper left panel) was also carried out on the extracts to show the interaction was specific. These experiments were also performed in MCF7 and Hela cells (Grb7 transfected), and similar results were observed (unpublished data). Right panels: the reverse process, that is, coimmunoprecipitation with resin-coupled Flna antibody was also performed (Lanes 1–2, upper right panel). The whole cell lysate (WCL, lower right panel), and the resulting immunocomplexes (IP: Filamin-a) were immunoblotted with Filamin-a and Grb7 antibodies. Non-specific IgG control and “non-antibody coupled beads” control coimmunoprecipitations (Lanes 3–4, upper right panel) show the interaction were specific. (COIP1: coimmunoprecipitation reaction1, COIP2: coimmunoprecipitation reaction 2, WCL1: whole cell lysate sample1, WCL2: whole cell lysate sample 2).

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Binding Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, In Vitro, Western Blot, In Vivo, Expressing, Immunoprecipitation, Transfection

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: The Grb10 RA-PH domain structure (Depetris et al., 2009, PDB# 3HK0). The left panel depicts the dimerized structure of the Grb10-RA-PH domains, with each RA and PH domains annotated. The right panel is an enlargement of the region outlined by dotted lines in the left panel. The Grb10 residues Y194 and Y341 (corresponding to Grb7 residues Y188 and Y338, respectively) are labeled for each RA-PH structure. The Grb10-RA-PH domain residues Q347 and N348, when mutated to alanine diminish dimerization, are marked by arrows.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Labeling

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: The Filamin-a interaction with Grb7 tyrosine mutants is decreased. Upper panels: Western blot analysis. HeLa Cells were transfected with pCMV-FLGrb7- Y188F (full-length Grb7-Y188F mutant), pCMV-FLGrb7-Y338F, and pCMV-FLGrb7- Y188F/Y338F (double mutant) constructs. Cells were lysed and immunoprecipitated by resin-coupled rabbit polyclonal anti-Grb7 (H-70), followed by Western blot analysis with Filamin-a antibody (3 F-180) and Grb7 antibody (H-70). Whole cell lysates were also probed with the same antibodies and anti-Actin(c-2) antibody. The figure shown is a representative blot of three independent experiments, each displaying similar results. Lower panel: the results of three different immunoprecipitation experiments were analyzed by densitometry. Filamin-a band densities were divided by corresponding Grb7 mutant band densities and were shown to be normalized to Grb7 WT. Standard deviations were calculated for each sample data set and plotted in bar-graph format.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Western Blot, Transfection, Mutagenesis, Construct, Immunoprecipitation

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: Grb7 and Filamin-a colocalize within SKBR3 cell membrane ruffles. A) SKBR3 Cells grown on 18 mm cover slips were fixed and probed for Grb7 (red), Filamin-a (green), filamentous actin (cyan) and DNA (blue). Colocalization was observed on the membrane ruffles at the apical domain of cells (Panels A–D, arrowheads), whereas no colocalization was observed on the ventral or basal surface (Panels E–H). Representative fluorescent images of the cells are shown, and example colocalization of Grb7 and Filamin-a is indicated by an arrowhead. Bar 10 μm. B) EGF stimulation results in Grb7/ Filamin-a colocalization to cell membrane ruffles. Serum starved SKBR3 cells treated for 30 min with 100 ng/ml EGF resulted in profuse cell membrane ruffling (Panel A–D), whereas serum starved, unstimulated cells lacked any detectable colocalization. Bar 10 μm.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques:

Journal: Journal of molecular recognition : JMR

Article Title: Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation

doi: 10.1002/jmr.2297

Figure Lengend Snippet: A) Grb7 knockdown significantly decreases wound closure rate in a wound scratch assay. SKBR3 cells were transfected with Grb7shRNA or Scramble shRNA. A wound was made with a 10 μl pipette tip across each well of a 12-well plate, and images were captured at different time points (in hours) as indicated. The invasion of wound-space over time was assessed by counting the number of cells present within the original wound boundaries at respective time points. The effect of Grb7shRNA on wound closure was compared with that of scramble shRNA treated cells. B) Quantification of the comparative decrease in Grb7 expression in SKBR3 cells upon either transfection with Grb7-specific shRNA or scrambled shRNA is shown in both Western Blot and densitometric bar-graph format.

Article Snippet: The following antibodies were obtained from Santa Cruz Biotechnology Inc, (Santa Cruz, CA): mouse monoclonal Filamin 1 (3 F180), mouse monoclonal Filamin 1 (PM6/317), rabbit polyclonal Grb7 (H-70), rabbit polyclonal Grb7 (C-20), mouse monoclonal GST (B-14), mouse monoclonal c-Myc (9E10), mouse monoclonal Actin (C-2), mouse monoclonal p-Tyr (PY20), goat anti-mouse IgG-HRP, and non-specific rabbit or mouse control IgG.

Techniques: Wound Healing Assay, Transfection, shRNA, Transferring, Expressing, Western Blot