Journal:

Article Title: Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3

doi: 10.1172/JCI200418046

Figure Lengend Snippet: siRNApyk2 decreases Pyk2 protein abundance in OKP cells. Typical blots. (A) Cells were transfected with 1.33 μg/well siRNApyk2 or siRNAGL2 (GL2 siRNA) or transfection reagent only (No siRNA) for 24 hours (at which time they were confluent), rendered quiescent for 24 hours, and harvested. FAK protein abundance was measured for comparison. (B) Cells were transfected with 1.33 μg/well siRNApyk2 for 24 hours (at which time they were confluent) and rendered quiescent. The length of quiescence varied from 0 to 48 hours, so that cells were harvested 24, 48, or 72 hours after the beginning of transfection. (C) Cells were transfected with the indicated amount of siRNApyk2 for 24 hours (at which time they were confluent), rendered quiescent for 24 hours, and harvested.

Article Snippet: All chemicals were obtained from Sigma-Aldrich with the following exceptions: penicillin and streptomycin (Whittaker MA Bioproducts); acetoxymethyl derivative of BCECF (Molecular Probes); TOPO TA Cloning kit, pcDNA3.1/HIS vector, ThermoScript RT-PCR System, Platinum Taq DNA Polymerase High Fidelity, and dNTP Mix (Invitrogen); pCI-neo, Tfx-50, BamHI , and EcoRI (Promega); DNA ligation kit (Takara); cell culture media, Lipofectamine Plus Reagent kits, and Superscript II kit (Gibco BRL); Trans IT-TKO siRNA Transfection Reagent (Mirus Corp.); anti-v-Src (Ab-1) monoclonal antibody, NP-40, and protein G-Plus agarose (Calbiochem); goat anti-Pyk2 polyclonal IgG antibody, rabbit anti-c-Src (SRC2) polyclonal IgG antibody, goat and rabbit anti-FAK polyclonal IgG antibodies, rabbit anti-Grb2 polyclonal IgG antibody, and anti-phosphotyrosine antibody (PY99) (Santa Cruz Biotechnology Inc.); rabbit anti-Pyk2 polyclonal IgG antibody (Upstate Biotechnology, Inc.); HRP–labeled anti-rabbit IgG and anti-mouse IgG (Amersham); and ECL kit and 32 P-γ-ATP (PerkinElmer).

Techniques: Quantitative Proteomics, Transfection, Comparison

Journal:

Article Title: Pyk2 activation is integral to acid stimulation of sodium/hydrogen exchanger 3

doi: 10.1172/JCI200418046

Figure Lengend Snippet: siRNApyk2 prevents NHE3 activation by acid. OKP cells were transiently transfected with 1.33 μg siRNApyk2 (pyk2 siRNA) per well for 24 hours (at which time they were confluent), rendered quiescent for 18 hours, and exposed to control (pH 7.4) or acid (pH 6.8) medium for 6 hours; then Na/H antiporter activity was assayed. Data are plotted as dpHi/dt, pH units/min. Controls were cells exposed to transfection reagent without siRNA (No siRNA) or cells transfected with a siRNA against a nonmammalian protein (GL2 siRNA); n = 7 (No siRNA), n = 6 (GL2 siRNA), and n = 10 (pyk2 siRNA). * P < 0.05 vs. pH 7.4.

Article Snippet: All chemicals were obtained from Sigma-Aldrich with the following exceptions: penicillin and streptomycin (Whittaker MA Bioproducts); acetoxymethyl derivative of BCECF (Molecular Probes); TOPO TA Cloning kit, pcDNA3.1/HIS vector, ThermoScript RT-PCR System, Platinum Taq DNA Polymerase High Fidelity, and dNTP Mix (Invitrogen); pCI-neo, Tfx-50, BamHI , and EcoRI (Promega); DNA ligation kit (Takara); cell culture media, Lipofectamine Plus Reagent kits, and Superscript II kit (Gibco BRL); Trans IT-TKO siRNA Transfection Reagent (Mirus Corp.); anti-v-Src (Ab-1) monoclonal antibody, NP-40, and protein G-Plus agarose (Calbiochem); goat anti-Pyk2 polyclonal IgG antibody, rabbit anti-c-Src (SRC2) polyclonal IgG antibody, goat and rabbit anti-FAK polyclonal IgG antibodies, rabbit anti-Grb2 polyclonal IgG antibody, and anti-phosphotyrosine antibody (PY99) (Santa Cruz Biotechnology Inc.); rabbit anti-Pyk2 polyclonal IgG antibody (Upstate Biotechnology, Inc.); HRP–labeled anti-rabbit IgG and anti-mouse IgG (Amersham); and ECL kit and 32 P-γ-ATP (PerkinElmer).

Techniques: Activation Assay, Transfection, Control, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Regulation of Vascular Endothelial Growth Factor Receptor 2-mediated Phosphorylation of Focal Adhesion Kinase by Heat Shock Protein 90 and Src Kinase Activities

doi: 10.1074/jbc.m405493200

Figure Lengend Snippet: FIG. 6. HSP90 regulates the VEGF-induced activation of RhoA and ROCK downstream of VEGFR2. A, quiescent HUVECs were pretreated for 60 min with geldanamycin (GA, 1 g/ml) or vehicle (0.25% Me2SO) or for 120 min with Y27632 (25 M). Cells were treated or not with VEGF (5 ng/ml for 15 min). Cells were extracted, and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of phospho-FAK Tyr407 (upper panel) and total FAK (lower panel). Data points represent means S.D. of two experiments. Representative blots are shown. B, quiescent HUVECs transiently transfected with an empty vector (EV) or with vectors expressing HA-FAK and dominant negative, kinase-defective mutant ROCK (KD4) or dominant negative mutant RhoA-N19 (N19) were treated or not with VEGF (5 ng/ml for 30 min). Cell extracts were prepared, and transfected HA-FAK was immunoprecipitated using an anti-HA mouse antibody. Control immunoprecipitation was done similarly using mouse IgG. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed to immunodetect phospho-FAK Tyr407 (upper panel) and immunoprecipitated HA-FAK to ensure equal loading and transfection efficacy (lower panel). C, quiescent HUVECs were pretreated for 60 min with geldanamycin (1 g/ml) or vehicle (0.25% Me2SO) and were treated or not with VEGF (5 ng/ml for 5 min). Proteins were extracted and incubated with GST-rhotekin to absorb activated RhoA. The samples were then separated by 12% SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted for RhoA (upper panel). The amount of total RhoA was monitored on the total extract (lower panel). Representative blots are shown. D–K, HUVECs plated on gelatin-coated chambers were left untreated (D and E) or were exposed to 5 ng/ml VEGF for 15 min (F and G) or were pretreated with Y27632 (25 M) for 2 h and then treated (H and I) or not (J and K) with 5 ng/ml VEGF for 15 min. F-actin (D, F, H, and J) was detected using fluorescein isothiocyanate-conjugated phalloidin. Vinculin (E, G, I, and K) was detected with specific antibody coupled with a biotin-labeled anti-mouse IgG and revealed with Texas Red-conjugated streptavidin. Cells were examined by confocal microscopy. Representative fields are shown. Similar results were obtained in three separate experiments. P-Y, phosphotyrosine; IP, immunoprecipitation.

Article Snippet: The rabbit polyclonal anti-c-Src antibody (SRC 2) and the mouse monoclonal antibody against RhoA (26C4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, SDS Page, Membrane, Immunodetection, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Mutagenesis, Immunoprecipitation, Control, Incubation, Labeling, Confocal Microscopy

Journal: Oncogene

Article Title: Suppression of autocrine cell proliferation and tumorigenesis of human melanoma cells and fibroblast growth factor transformed fibroblasts by a kinase-deficient FGF receptor 1: evidence for the involvement of Src-family kinases.

doi: 10.1038/sj.onc.1201159

Figure Lengend Snippet: Figure 6 Src-family kinase activity is modulated by FGFR1 activity. Histograms in (a) and (b) show Src-2 immune complex kinase activity (recognizing Src, Yes and Fyn) employing (500 mg protein/precipitation) and Raytide as an exogenous substrate. (a) Kinase activity in normal melanocytes. Assays were performed with lysates from non-stimulated cells (N/A), or from melanocytes that have been stimulated for 15 min with bFGF or HGF/SF (as indicated). (b) Kinase activity in parental and transduced melanoma cells. Lysates from unstimulated melanoma cells were used. Notice that data in (a) and (b) were plotted using dierent scales. (c) Expression of Src-family kinases. Whole cell lysates (40 mg proteins/lane) were subjected to Western immunoblotting with (from top to bottom) Src-2, ac-Src (N-16, Santa Cruz), ac- Yes, or rabbit antiserum to c-Fyn (UBI). Normal human melanocytes (NM) and transduced 586 melanoma cells are as indicated. Position of size marker is indicated in kDa the left hand side

Article Snippet: The following antibodies were used: Monoclonal antibody 4G10 against phosphotyrosine (aPY; UBI, Upstate Biotechnology, Inc., Lake Placid, NY); Src-2-rabbit polyclonal antibody raised against a peptide corresponding to amino acids 509 ± 533 mapping at the carboxy terminus of Src p60 (Santa Cruz Biotechnology, Santa Cruz, CA), reactive with p60 c-Src, p62 c-Yes and p59 c-Fyn; anti-c-Src mAb 327 (a gift from Dr Joan Brugge, ARIAD Pharmaceuticals, Inc, Cambridge, MA) and polyclonal antibodies N-16 (Santa Cruz); anti-c-Yes mAb (Clone 1, Transduction Laboratories, Lexington, KY); anti-Fyn mAb [fyn(15) Santa Cruz Biotechnology], or rabbit polyclonal antibodies (FYN3; Santa Cruz), or rabbit antiserum raised against a synthetic peptide comprising amino acid residues 35 ± 51 of human Fyn (UBI).

Techniques: Activity Assay, Immune Complex Kinase Assay, Expressing, Western Blot, Marker