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IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting <t>with</t> <t>anti-GFP</t> antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).
Mouse Monoclonal Anti Gfp Antibody (Jl 8, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting <t>with</t> <t>anti-GFP</t> antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).
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IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting <t>with</t> <t>anti-GFP</t> antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).
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IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting <t>with</t> <t>anti-GFP</t> antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).
Anti Gfp Ab, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting with anti-GFP antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).

Journal:

Article Title: Rho overexpression leads to mitosis-associated detachment of cells from epithelial sheets: A link to the mechanism of cancer dissemination

doi: 10.1073/pnas.0404723101

Figure Lengend Snippet: IAR-2 epitheliocytes stably express functional constitutively active RhoA (EGEP-RhoA Q63L) protein. RhoA expression was confirmed by fluorescence in live cells (A) and by immunoblotting total cell proteins (B). (A) An island of cloned IAR-2 epithelial cells stably expressing EGFP-RhoA Q63L. This same island of cells is shown by phase-contrast microscopy in Fig. 2C. (Bar = 50 μm.) (B) RhoA pull-down assay examining activity of RhoA construct. Transferred cells were lysed, and EGFP-RhoA Q63L was affinity precipitated by using recombinant GST-Rhotek-PBD (RBD-GST) bound to latex beads. EGFP-RhoA Q63L bound to RBD-GST and the expression of EGFP-RhoA Q63L were analyzed by Western blotting with anti-GFP antibodies. IAR-2 cell stably expressed EGFP (control) or EGFP-RhoA Q63L (lysate). EGFP-RhoA Q63L was bound to GST-RBD (RBD-GST), indicating the expressed construct was functionally active. EGFP-RhoA Q63L did not bind to GST beads (GST).

Article Snippet: The resolved proteins were transferred to nitrocellulose and probed with mouse monoclonal anti-GFP antibody (JL-8) (BD Biosciences, Clontech; dilution 1:1,000).

Techniques: Stable Transfection, Functional Assay, Expressing, Fluorescence, Western Blot, Clone Assay, Microscopy, Pull Down Assay, Activity Assay, Construct, Recombinant