Journal: bioRxiv

Article Title: Fibrin fragment E potentiates TGF-β-induced myofibroblast activation and recruitment

doi: 10.1101/829945

Figure Lengend Snippet: Interactions between soluble recombinant αVβ 3 (5-50 nM) and immobilized FnE (A) or FnDD (B) were determined by SPR. Immobilization was at 952 RU and 1251 RU respectively. (C) SPR sensorgrams of the interaction between FnE and integrin αVβ 3 fitted to a bivalent analyte model (i), a heterogeneous ligand model (ii) or to the 1:1 two state interaction model (iii). (D) Representative western blot and (E) quantification of α-SMA protein levels of HFL1 fibroblasts in the presence of the cyclic-RGD peptide EMD 66203 in combination with TGF-β (5 ng/ml) and/or FnE (2 nM). α-SMA was normalized against β-actin and expressed as compared to vehicle (DMSO) treated controls. (F) Chemotaxis and (G) net migration of HFL1 cells in presence of 5 µM cyclic-RGD peptide. Dashed grey lines indicate mean values for cells without addition of cyclic-RGD. (H) Integrin β3 and β5 protein levels in HFL1 cells after transfection with siRNA selectively targeting these integrins, as indicated. (I) α-SMA ( ACTA2 ) mRNA levels of siRNA transfected cells stimulated with TGF-β (5 ng/ml) with or without addition of FnE (2 nM) for 48 h. (J) Chemotaxis and (K) net migration of HFL1 cells treated with siRNA to suppress integrin β3 or β5 in response to a PDGF-BB (0-20 ng/ml) or FnE (0-2 nM) gradients. * p<0.05. Error bars represent standard error of the mean ( n ≥3).

Article Snippet: Primary antibodies used were mouse anti-α-SMA (clone 1A4, Sigma-Aldrich) 1:10 000, rabbit anti-β-actin (ab8227, Abcam) 1:5000, mouse anti-PARP1 (BD-bioscience) 1:1500, mouse anti-integrin β3 (Cell signaling) 1:500, rabbit anti-integrin β5 (Abnova) 1:250, all added in blocking buffer with 0.1% Tween-20.

Techniques: Recombinant, Western Blot, Chemotaxis Assay, Migration, Transfection

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Fluorescence confocal images showing A488-irisin binding in HEK293T cells. HEK293T cells were either transiently transfected with control vector or full-length αV and β5 plasmids. 2 nM Hsp90α was used for 1 hr pretreatment, and 2 nM A488-irisin-His was subsequently used for 5 min treatment. Scale bar: 20 μm. (B) Anti-phosphorylated FAK (Y397) and anti-FAK western blots showing the levels of integrin signaling upon irisin and/or Hsp90α treatments. HEK293T cells were transfected and treated in the same way as (A), except for the addition of the shown amounts of unlabeled irisin-His (0.1 nM or 1 nM) and Hsp90α (1 nM). Anti-αV and anti-β5 antibodies were used to probe the levels of the ectopically expressed αV and β5. (C) Immunofluorescence confocal images showing cell surface Hsp90α in SK-Mel2 cells. Live cells were treated with either control IgG or anti-Hsp90α at 4°C. Scale bar: 50 μm. (D) Quantification of the percentage of SK-Mel2 cells expressing cell surface Hsp90α in (C) (significant if p-value < 0.05 by unpaired t-test). (E) Fluorescence confocal images showing A647-irisin binding in SK-Mel2 cells. Live cells were pretreated with either control IgG or anti-Hsp90α at 4°C for 1 hr followed by 2 nM A647-irisin-His treatment at room temperature for 5 min. Scale bar: 50 μm. (F) Quantification of the percentage of A647-positive cells in (E) (significant if p-value < 0.05 by unpaired t-test). (G) Co-immunoprecipitation assay of endogenous cell surface αV and β5 using SK-Mel2 cells. Endogenous cell surface Hsp90α was captured by anti-Hsp90α in live cells at 4°C. (H) Crystal violet assay showing does-dependent inhibition of the cell viability of SK-Mel2 upon irisin treatment. Grey bar: control treatment with PBS. Concentrations of irisin-His used for the treatments were indicated (one-way ANOVA). (I) Crystal violet assay showing the inhibition of irisin-mediated effect in SK-Mel2 cells by anti-Hsp90α or control antibody. Grey bar: control treatment with PBS. 50 ng/mL of irisin-His was used (one-way ANOVA). (J) Western blot of mouse inguinal fat tissue lysates using the indicated antibodies to probe integrin signaling. Mice were given anti-Hsp90α antibody or control IgG (500 μg/kg) subcutaneously 24 hrs before a bolus injection of recombinant irisin (5 mg/kg) directly into the inguinal fat pads. The mice were sacrificed and inguinal fat tissues were harvested 20 min after irisin injection.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Fluorescence, Binding Assay, Transfection, Control, Plasmid Preparation, Western Blot, Immunofluorescence, Expressing, IF-P, Co-Immunoprecipitation Assay, Crystal Violet Assay, Inhibition, Injection, Recombinant

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: (A) Flow charts of the steps used in three different methods for analyzing αVβ5-Apo and αVβ5/Hsp90α cryo-EM samples. (B) 2D classes (generated by method 1) of αVβ5 particles in each of the three conformational states and the numbers (quantified by all three methods) of particles in each state. (C) Quantification of the percentage of distinguished particles (“likely open” particles were not included) in each of the three conformational states. (D) Fluorescence anisotropy assay for A488-irisin binding by αVβ5, the αVβ5/Hsp90α complex in the presence of 1 mM MgCl2 and 1 mM CaCl2, or αVβ5 in the presence of 1 mM MnCl2. 50 nM A488-irisin-His was used in the assay. (E) Cartoon diagram showing a two-step process of the irisin action through αVβ5. Irisin alone has low affinity for the closed-state αVβ5. Hsp90α, Mn2+ ion, or other possible factors, “opens” αVβ5, allowing for high-affinity irisin binding and effective signaling transduction through its integrin receptor. (F) and (G) TALON pull-downs performed using 1 μM bead-bound clasped and tagged αVβ5. These were mixed with 2 μM untagged Hsp90α without bound nucleotide (Hsp90α-Apo) or Hsp90α charged with the indicated nucleotides (F), or Hsp90α nonhydrolyzing mutant (G95D) (G), and bound samples were analyzed by Coomassie staining and anti-Hsp90α western blot.

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Cryo-EM Sample Prep, Generated, Fluorescence, Binding Assay, Transduction, Mutagenesis, Staining, Western Blot

Journal: Molecular cell

Article Title: Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α

doi: 10.1016/j.molcel.2023.05.008

Figure Lengend Snippet: Figure 5C

Article Snippet: Rabbit Monoclonal Anti- Integrin β5 , Abcam , Cat. # ab184312.

Techniques: Control, Virus, Recombinant, Expressing, Protease Inhibitor, Lysis, Transfection, Electron Microscopy, Immunodepletion, Clone Assay, Protein Purification, Endotoxin Assay, Bradford Assay, Staining, Mass Spectrometry, Mutagenesis, Software, Isolation

Journal: The Journal of Neuroscience

Article Title: Integrins Control Dendritic Spine Plasticity in Hippocampal Neurons through NMDA Receptor and Ca 2+ /Calmodulin-Dependent Protein Kinase II-Mediated Actin Reorganization

doi: 10.1523/JNEUROSCI.4091-05.2006

Figure Lengend Snippet: Integrin subunits β1, β3, and β5 are localized in synapses of hippocampal neurons. A, B, Immunofluorescent labeling of 14 DIV hippocampal neurons showed localization of β3 (red; A) and β5 (red; B) in close proximity to synaptophysin-positive presynaptic boutons (green). Arrows indicate colocalization of integrins (red) and synaptophysin (green). Scale bars: top panel, 10 μm; bottom panel, 5 μm. C, Coimmunolabeling of β3 integrin clusters (red) and postsynaptic scaffold protein PSD-95 (green) in 14 DIV hippocampal neurons. β3-Immunopositive puncta (red) colocalized with PSD-95 (green). Arrows point to examples of synaptic integrin clusters. Scale bars: top panel, 10 μm; bottom panel, 5 μm. D, Immunofluorescent labeling of β3 integrin (red) in GFP-expressing 14 DIV hippocampal neurons. Hippocampal neurons were transfected with GFP at 5 DIV. At 14 DIV, hippocampal neurons were immunolabeled with anti-β3 integrin antibody. Arrows point to examples of β3 integrin clusters (red) that are localized in dendritic spines (green). Scale bars: top panel, 10 μm; bottom panel, 5 μm. E, F, Immunofluorescent labeling of 14 DIV hippocampal neurons shows localization of β3 (green; E) and β1 (green; F) in close proximity to polymerized actin (F-actin) that is labeled with rhodamine-coupled phalloidin (red). Arrows indicate colocalization of integrins (green) and F-actin (red). Scale bars: top panel, 10 μm; bottom panel, 5 μm. G, Western blot analysis of the subcellular distribution of β3 and β5 integrins in adult mouse hippocampus. Subcellular fractions were prepared from adult mouse hippocampus as described in Materials and Methods. Equal amounts (12 μg) of protein from each fraction were resolved on an 8–16% Tris-glycine gels and immunoblotted with specific antibodies against β3, β5, NR2A/B, and PSD-95. H, Homogenate; P2, crude synaptosome fraction; LP1, presynaptic and postsynaptic membrane fraction.

Article Snippet: The primary antibodies used were the following: mouse anti-PSD-95 (33 μg/ml; clone 6G6; Affinity BioReagents, Golden, CO), rabbit anti-NMDAR2A/B (1 μg/ml; AB1548; Chemicon), rabbit anti-β3 integrin and rabbit anti-β5 integrin (20 μg/ml; AB1932 and AB1926; Chemicon), mouse anti-β1 integrin (20 μg/ml; MAB1987Z; Chemicon), mouse anti-MAP2 (5 μg/ml; clone HM-2; Sigma), rabbit anti-GAD (1 μg/ml; GAD-65; Chemicon), and mouse anti-synaptophysin (61 μg/ml; SVP-38; Sigma).

Techniques: Labeling, Expressing, Transfection, Immunolabeling, Western Blot

Journal: The Journal of Neuroscience

Article Title: Integrins Control Dendritic Spine Plasticity in Hippocampal Neurons through NMDA Receptor and Ca 2+ /Calmodulin-Dependent Protein Kinase II-Mediated Actin Reorganization

doi: 10.1523/JNEUROSCI.4091-05.2006

Figure Lengend Snippet: Function-blocking antibodies against β1 and β3 integrin partially blocked RGD-induced actin reorganization and dendritic spine elongation. A, Quantitative analysis of the number of F-actin, synaptophysin, and actin/synaptophysin double-positive clusters per 10 μm of dendrite. Data represent the average number of clusters per 10 μm of dendrite. Error bars indicate SEM (n = 10 neurons per group). **p < 0.01; ***p < 0.001 by one-way ANOVA. B, C, Quantification of dendritic protrusion length (B) and number (C) in 14 DIV GFP-labeled hippocampal neurons after treatment with RAD, RGD, RGD plus anti-β1 antibody, RGD plus anti-β3 antibody, or RGD plus anti-β1 and anti-β3 antibodies. Data represent the mean protrusion length (B) or average number of protrusions per 10 μm of dendrite (C). Error bars indicate SEM (n = 10 neurons per group). ***p < 0.001 by one-way ANOVA.

Article Snippet: The primary antibodies used were the following: mouse anti-PSD-95 (33 μg/ml; clone 6G6; Affinity BioReagents, Golden, CO), rabbit anti-NMDAR2A/B (1 μg/ml; AB1548; Chemicon), rabbit anti-β3 integrin and rabbit anti-β5 integrin (20 μg/ml; AB1932 and AB1926; Chemicon), mouse anti-β1 integrin (20 μg/ml; MAB1987Z; Chemicon), mouse anti-MAP2 (5 μg/ml; clone HM-2; Sigma), rabbit anti-GAD (1 μg/ml; GAD-65; Chemicon), and mouse anti-synaptophysin (61 μg/ml; SVP-38; Sigma).

Techniques: Blocking Assay, Labeling

Journal: Nature protocols

Article Title: Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

doi: 10.1038/nprot.2015.122

Figure Lengend Snippet: Antibodies and small-molecule stains validated for cell-phenotyping in PACT- and PARS-processed tissues.

Article Snippet: Rabbit anti–integrin-β4 and anti-integrin β5 IgGs , Santa Cruz Biotechnology (β4: sc-9090, β5: sc-14010); 1:200 dilution.

Techniques: Formulation, Staining, Plasmid Preparation, Purification, Labeling, Hybridization

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: Identified interactors with human integrin β 5 subunit cytoplasmic domain by yeast two-hybrid screening

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Clone Assay

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: PAK4 interacts with the integrin β 5 subunit. (A) Amino acid sequence comparison of mPAK4 KD, found by the yeast two-hybrid screening with hPAK4, Drosophila MBT (MBT), and hPAK1 KDs. Dashes stand for identical amino acids and the variations of amino acids are indicated by letters in hPAK4, MBT, and hPAK1 sequences. The mPAK4 KD is numbered according to that of hPAK4 . (B) PAK4 association with integrin β5 in GST pull-down assays. GST–PAK4 KD fusion protein pulled down endogenous integrin β5 in cell lysates (top panel). GST–β5 cytoplasmic domain fusion protein pulled down overexpressed HA–PAK4 (bottom). The positive controls represent Western blot of the input lysates for marking the size and showing the presence of integrin β5 and PAK4. (C) PAK4 association with integrins in living cells. (C, top) Anti–integrin β3 mAb AP3 and anti–integrin αvβ5 mAb P1F6 coimmunoprecipitation of PAK4 in COS-7 cells expressing HA–PAK4. (C, middle and bottom) An anti-HA mAb coimmunoprecipitated HA–PAK4 with both αv and β5 integrin subunits. The positive controls represent Western blot of the input lysates for marking the size and showing the presence of integrins and PAK4. (D) Association of endogenous PAK4 with endogenous integrin αvβ5 in MCF-7 cells analyzed by IP. IP using rabbit IgG or α-Rab1 are negative controls.

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Sequencing, Two Hybrid Screening, Western Blot, Expressing

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: Mapping of the PAK4 binding region within the integrin β 5 cytoplasmic domain. (A) Mapping of the PAK4-binding region in the integrin β5 cytoplasmic domain. Various regions of the integrin β5 cytoplasmic domain were cloned into the bait vector pEG202 and then mated with PAK4 KD in the prey vector in a yeast two-hybrid assay. The PAK4 binding region was mapped to aa 759–767 of the β5 cytoplasmic domain. (B) Association of endogenous PAK4 with the membrane-proximal region within integrin β5 cytoplasmic domain was examined by a GST pull-down assay, including GST fused to a β5 deletion mutant lacking the PAK4-binding region mapped by yeast two-hybrid analysis. (C) Sequence comparison of the PAK4-binding region of integrin β5 with other integrin cytoplasmic domains (top). The Rack1- and PAK4-binding regions within integrin β5 are indicated (bottom).

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Y2H Assay, Pull Down Assay, Mutagenesis, Sequencing

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: Mapping of the integrin β 5–binding region within the PAK4 KD. (A) Various regions of the PAK4 KD were cloned into the prey vector pJG4-5 and then mated in a yeast two-hybrid assay with the integrin β5 cytoplasmic domain in the bait vector. Amino acids 505–530 in the PAK4 KD were identified as the responsible region for interaction with the integrin β5 cytoplasmic domain. Therefore, PAK4 aa 505–530 is designated as the IBD. (B) PAK4-ΔIBD with deletion of aa 505–530 was incapable to associate with integrin αvβ5 in mammalian cells, as examined by an IP analysis. Lower panel shows expression levels of PAK4 and PAK4-ΔIBD in lysates used for IP. (C) The corresponding sequences in other PAK family members are compared with PAK4 IBD with identical amino acids in bold. (D) Schematic illustration of the PAK4 structure, including the Cdc42/Rac interactive domain ATP-binding domain, IBD, and KD within PAK4.

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Y2H Assay, Expressing

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: Relocalization of PAK4 to the cell membrane and colocalization of PAK4 and integrin α v β 5 in MCF-7 cells. (A) MCF-7 human breast carcinoma cells, under normal culture conditions or replated onto VN or PLL for indicated times, were stained for endogenous PAK4 using an anti-PAK4 pAb (green), for actin using phalloidin–rhodamine (red), and for nuclei by Hoechst (blue). Arrowheads indicate PAK4 localized to lamellipodia or ruffles after replating onto VN. (B) MCF-7 cells were replated onto VN and costained for endogenous PAK4 (green) and endogenous integrin αvβ5 (red). In the top panel (30 min after replating), integrin αvβ5 was found at lamellipodia (arrowheads). In the bottom panel (60 min after replating), integrin αvβ5 has started to form focal complexes at lamellipodia where it colocalized with PAK4 (arrowheads). Bars, 10 μm.

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: PAK4 translocation to lamellipodia by integrin ligation to VN does not depend on Cdc42 binding, integrin interaction, or PAK4 kinase activity. (A) Flag-tagged PAK4 mutants used for translocation studies. PAK4-L19, 22 lacks binding capacity to Cdc42/Rac. PAK4-M350 and PAK4-ΔIBD are both kinase dead, and PAK4-ΔIBD cannot bind integrin β5. (B) M21 cells were transfected with Flag-tagged wt PAK4, PAK4 mutants, or the control Flag-BAP vector. Cells were stained using an anti-Flag mAb (green), and stained for actin using phalloidin–rhodamine (red) and for nuclei using Hoechst (blue) before (B) or after (C) replating onto VN for 1 h. Arrows indicate the distribution in lamellipodia of PAK4 and PAK4 mutants. Bars, 20 μm. (D) Quantification of membrane-localized wt PAK4 or PAK4 mutants before and after replating onto VN for 1 h. Bars represent percent of cells with membrane-localized wt PAK4 or PAK4 mutants of the total cells counted and are expressed as mean ± SD. In a statistical evaluation comparing before and after cell replating onto VN, all PAK4 variants gave P < 0.05 (*) or P < 0.01 (**) by a paired t test. PAK4-L19, 22 localization to the membrane was also higher than wt PAK4 under normal culture conditions (P < 0.05 [*]).

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Translocation Assay, Ligation, Binding Assay, Activity Assay, Transfection, Plasmid Preparation, Staining

Journal: The Journal of Cell Biology

Article Title: p21-activated kinase 4 interacts with integrin αvβ5 and regulates αvβ5-mediated cell migration

doi: 10.1083/jcb.200207008

Figure Lengend Snippet: PAK4 stimulates integrin α v β 5–mediated cell migration. (A) MCF-7 cells transiently transfected with EGFP–PAK4 or EGFP control were analyzed for haptotactic cell migration toward VN in the presence or absence of normal mouse IgG and functional blocking mAbs LM609 (anti-αvβ3) or P1F6 (anti-αvβ5) (left panel) and toward collagen type I (right panel). Data represent the average of three independent experiments and were normalized to the transfection efficiency of individual vectors, as determined by flow cytometry. Statistical evaluation comparing EGFP to EGFP–PAK4 on VN gave P < 0.05 by t test. (B) Cell migration analyzed as in A of MCF-7 cells stably expressing EGFP–PAK4 or EGFP. All results are expressed as mean values ± SEM of three independent experiments using triplicate analysis in each experiment. Statistical evaluation by t test gave P < 0.05 for EGFP–PAK4 compared with EGFP on VN. (C) Overexpression of PAK4 decreases cell adhesion on VN. Cell attachment of MCF-7 cells stably expressing EGFP–PAK4 or EGFP at different coating concentrations of VN was determined. (D) Transient EGFP–PAK4 expression does not change the membrane expression levels of integrin αvβ5 in MCF-7 cells as measured by flow cytometry. αvβ5 expression was plotted versus EGFP content and the αvβ5 cell surface levels in EGFP-transfected cells (bottom left) was compared with that in EGFP–PAK4 cells (bottom right). Values indicate the mean fluorescence of αvβ5 staining in EGFP-positive cells. Unstained MCF-7 cells (top left) and staining without primary mAb (top right) were used to determine specificity and background.

Article Snippet: Approximately 500 μg of precleared lysates were immunoprecipitated by 4 μg rabbit anti-HA pAb Y11 (Santa Cruz Biotechnology, Inc.), anti-αvβ5 mAb P1F6 (Life Technologies), or anti-β3 mAb AP3 (GTI) and were separated and probed with rabbit anti–human integrin β5 cytoplasmic pAb, anti–integrin αv pAb (Chemicon), or anti–HA tag mAb F-7 (Santa Cruz Biotechnology, Inc.) and then with an HRP-conjugated secondary pAb (Jackson ImmunoResearch Laboratories) and visualized by ECL technique as previously described ( ).

Techniques: Migration, Transfection, Functional Assay, Blocking Assay, Flow Cytometry, Stable Transfection, Expressing, Over Expression, Cell Attachment Assay, Fluorescence, Staining