function-blocking antibodies against the integrin subunits β 1 (6s6)  (Millipore)

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Antibodies, commercial sources, and applications.

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Migration, Microscopy, Western Blot, Purification, Immunofluorescence

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Primers used for the amplification of integrin cDNA from rat fetal brain neurons.

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Amplification

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Effects of antibodies, calcium modulators, and pharmacological inhibitors on neuritogenesis. (a) Representative images showing neuritogenesis in the absence (control) or presence of control IgG (50 nMole), antibodies against β 1 (50 or 100 nMole) or α 3 (50 or 100 nMole) integrin subunits on laminin. BAPTA-AM at low concentration (2.5 μ M) inhibited neuritogenesis and at higher concentration (10 μ M) totally abolished neuritogenesis. Bar150 microns. (b) Mean + standard error of mean values of neurite lengths/image in untreated neurons (1) and those treated with control IgG, monoclonal antibody against β 1 or α 3 integrin subunit at 50 nMole or 100 nMole concentration. Neurite lengths reduced significantly ( * P < 0.05) in neurons treated with antibody against β 1 integrin subunit at both 50 nMole (2) and 100 nMole (3) concentrations compared to control (1). Neurons treated with monoclonal antibody against α 3 integrin subunit at 50 nMole (4) were lower than the control (1) but was not statistically significant ( P > 0.05). Neurons treated with monoclonal antibody against α 3 integrin subunit at 100 nMole (5) significantly inhibited the neuritogenesis ( * P < 0.055). Neuritogenesis was not altered in presence of 50 (6) or 100 nMole (7) control IgG ( P > 0.05). (c) Mean + standard error of mean values of neurite lengths/image of neurons treated with PP2, PP3, U2, U3, 2-APB, activated Calphostin, BAPTA-AM (2.5 μ M), and PD for 22 h in culture (filled bars) were significantly different ( * P < 0.05) from untreated neurons (blank bar) and negative controls (PP3 or U3) (filled bars). No significant changes in neurite lengths ( P > 0.05) per image were recorded in neurons treated with RR.

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Concentration Assay

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Transcripts of integrin subunits in the fetal cortical neurons. Ethidium bromide stained PCR products after agarose gel electrophoresis. Target mRNA species are shown at the bottom of the image. The 600 bp band of the 100 bp marker (M) is shown by arrows heads on sides.

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Staining, Agarose Gel Electrophoresis, Marker

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Effects of antibodies on the migration of fetal cortical neurons. Monoclonal antibodies (shown below) against β 1 and α 3 integrin subunits significantly ( * P < 0.05) inhibited the migration of neurons (Neurons/field) on membranes coated with laminin (10 μ g/mL) (a). The migrations of neurons on laminin-coated membranes were not significantly ( P > 0.05) altered by antibody against α 6 or αv subunit (the negative control) and control antibodies (IgG or IgM). The migrations of neurons on fibronectin (100 μ g/mL) coated membranes were significantly ( * P < 0.05) inhibited by the antibody against β 1 integrin subunit only (b). The migrations of neurons on fibronectin-coated membranes were not altered by control antibodies (IgG or IgM) at P > 0.05.

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Migration, Negative Control

Journal: Neuroscience Journal

Article Title: Roles of Integrins and Intracellular Molecules in the Migration and Neuritogenesis of Fetal Cortical Neurons: MEK Regulates Only the Neuritogenesis

doi: 10.1155/2013/859257

Figure Lengend Snippet: Schematic of integrin signaling cascade and its perturbation with antibodies and pharmacological agents. Integrin subunits (red and green bars on top) are shown intercalated in the membrane and interacting with ECM molecule (red and green horizontal lines). Molecules involved in signaling events are labeled and arrows point to the directions of signaling that starts with the engagement of integrin subunit with the extracellular matrix. Directions of calcium mediated signaling are shown by dashed green arrows. Inhibitors (see ) used for blocking signaling molecules and paths are shown by red blocks. Cross-talk between integrin and GF signaling is shown by double headed horizontal arrow close to membrane. Mab: monoclonal antibody, GF: growth factor, RTK: receptor tyrosine kinase, MEK: MAP kinase kinase, PLC: phospholipase C, PIP2: phosphotidal inositol biphosphate, IP3: inositol (1,4,5)-triphosphate (IP3), DAG: diacylglycerol and PKC: protein kinase C, Ras: G protein, Raf: MAPKKK, MEK: MAPKK, ERK: extracellular signal regulated kinase (MAPK).

Article Snippet: Monoclonal antibodies against β 1 integrin subunit (Clone Ha2/5) , BD Biosciences (San Jose, CA) , Migration assays.

Techniques: Labeling, Blocking Assay

Journal: bioRxiv

Article Title: THE ASSOCIATION OF CD47 WITH INTEGRIN Mac-1 REGULATES MACROPHAGE RESPONSES BY STABILIZING THE EXTENDED INTEGRIN CONFORMATION

doi: 10.1101/2022.09.30.510402

Figure Lengend Snippet: (A) Purified inflammatory peritoneal mouse macrophages were immunoprecipitated with anti-α M rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the α M or CD47. Blots of the total cell lysates of WT and CD47-deficient macrophages using rabbit polyclonal antibody against the α M or CD47are shown in the left panel. M, molecular weight markers. The molecular weight of the α M (165 kDa) and β 2 (95 kDa) integrin subunits and CD47 (47 kDa) are indicated on the right of the panel. (B) Murine IC-21 macrophages were immunoprecipitated with anti-α M rabbit polyclonal antibody or rabbit polyclonal anti-CD47 antibody, and blots were analyzed with rabbit polyclonal antibody against the α M or CD47. (C) Biotinylated Mac-1-HEK293 cells were lysed and immunoprecipitated with mAb 44a against the α M subunit or isotype control IgG1. Blots were disclosed with streptavidin-alkaline phosphatase (AP). (D) Suspended (denoted “s”) or adherent (denoted “a”) Mac-1-HEK293 cells were lysed and immunoprecipitated with anti-α M mAb 44a or anti-β 2 mAb IB4. Blots were analyzed with anti-α M , anti-β 2 , and anti-CD47 antibodies. (E) The ratios of CD47 to the α M and β 2 integrin subunits in the immunoprecipitates from suspended and adherent cells were determined from the densitometry analyses of blots. The ratio of CD47 to each integrin subunit in suspended cells was taken as 1.0. (F) Lysates of biotinylated Mac-1-HEK293 cells were immunoprecipitated with anti-CD47 mAb B6H12; then immunoprecipitates were subjected to Western blotting probed with streptavidin-AP ( left panel; 1 IP). After the first round of immunoprecipitation, the supernatant was immunoprecipitated with anti-α M mAb 44a ( middle panel; 2 IP). The third round of immunoprecipitation (3 IP) was performed using anti-β 1 mAb ( right panel ).

Article Snippet: The mouse anti-human α M mAb (catalog #66519-1-Ig) and the rabbit polyclonal antibody, which recognizes both mouse and human β 1 integrin subunits (catalog #12594-1-AP), were from Proteintech (Rosemont, IL).

Techniques: Purification, Immunoprecipitation, Molecular Weight, Western Blot

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Conformational changes in the α β 1 integrin headpiece. ( a ) Part of the α β 1 integrin headpiece in cartoon representation. Helices α1 and α7 are highlighted in orange and blue. The propeller-βA distance is measured between the respective centers of mass (pink circles). Colors of the domains are according to Supplementary Fig. . ( b ) Close-up view of the βA domain with the docked TUDC structure (stick representation) . This complex structure was used to generate other starting structures by modifying the bile acid. Angles measured during the course of the MD simulations: orange: α1 kink angle; blue: α7 tilt angle. Mg 2+ ions are depicted as red spheres; the one at the MIDAS site is labeled M, the one at the ADMIDAS A. ( c–h ) α1 kink angle (orange), α7 tilt angle (blue), and propeller-βA distance (pink) during the course of three (color shades) MD simulations of each of the complexes between α β 1 integrin and ( c ) TUDC, ( d ) nor UDCA, ( e ) T nor UDCA, ( f ) GUDC, ( g ) UDCA, and ( h ) TC. For clarity, the time course data (left) has been smoothed by Bezier curves. Relative frequencies of the parameters (right) are calculated for the last 100 ns of each simulation. The frequency distributions have been overlaid with Gaussians according to their means and standard deviations (black curves).

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Labeling

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Activation of α β 1 integrins in MD simulations compared to activation of α IIb β 3 integrin in crystal structures. ( a ) Structural overlay of the βA domain (transparent: starting structure; opaque: closest-to-average structure from the last 100 ns) by fitting on the β-propeller domain . Pink arrows denote the positional shift of the βA domain relative to the β-propeller domain, resulting in an increased propeller-βA domain distance. ( b ) Overlay of the closed (lighter colors; PDB ID 3FCS) and open (darker colors; PDB ID 3FCU) conformations of the βA domain in α IIb β 3 integrins. Straightening of the α1 helix (orange) and tilting of the α7 helix (blue) are indicated by white arcs and bars. ( c ) Average of the α1 kink angle (yellow), α7 tilt angle (blue), and β-propeller – βA-domain distance (magenta) over three replicates of MD simulations versus the rank of the bile acids according to their agonist activity towards α β 1 integrin as observed in Fig. and ref. . Dashed lines represent correlation lines; fit parameters are given in the figures. Vertical lines separate the dataset into inactive (left), weakly active (middle), and highly active (right) bile acids.

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Effect of nor UDCA, T nor UDCA, GUDC, and UDCA on β 1 integrin activation. Rat livers were perfused with ( a ) nor UDCA, ( b ) T nor UDCA, ( c ) GUDC, and ( d ) UDCA for up to 60 min with the concentrations indicated. Liver samples were immunostained for the active conformation of β 1 integrin (red). The scale bar corresponds to 50 µm. Representative pictures of at least three independent experiments are depicted. To enhance visibility of the images, the white point of all channels in the RGB color space was reduced from the standard value of 255 to a value of 128. For each image, pixel intensities are indicated as average ± SEM. Nor UDCA and T nor UDCA triggered activation of the β 1 integrin subunit within 15 min, with stronger effects observed with nor UDCA. In contrast, equimolar concentrations of UDCA and GUDC were ineffective. Like TUDC (Fig. ) , nor UDCA-induced β 1 integrin activation occurred primarily in the intracellular compartment of hepatocytes. ( e ) Staining of total α β 1 integrin (red) and filamentous actin labeled with FITC-coupled phalloidin (green) at t = 0 min and t = 15 min after perfusion with nor UDCA, TUDC, GUDC, and UDCA.

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Activation Assay, Staining, Labeling

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Immunofluorescence staining and quantification of β 1 integrin. ( a ) Rat livers were perfused with either nor UDCA or TUDC (20 µmol/l each) for up to 15 min and immunostained for the active β 1 integrin conformation and actin as given under “Experimental Procedures”. The scale bar corresponds to 20 µm. Representative pictures of three independent experiments are depicted. ( b ) β 1 integrin fluorescence was quantified by using ImageJ analysis software. Whereas TUDC induced β 1 integrin activation within 5 min, nor UDCA activated β 1 integrins with lower effect. * p < 0.05 denotes statistical significance compared with the unstimulated control; # p < 0.05 statistical significance between nor UDCA and TUDC perfusion.

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Immunofluorescence, Staining, Fluorescence, Software, Activation Assay

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Affinities of TUDC and nor UDCA and the control peptide towards the RGD-recognizing integrin α 5 β 1 obtained from an ELISA-like solid-phase binding assay.

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: nor UDCA-induced activation of Erk-1/-2, p38 MAPK and Src. Rat livers were perfused with nor UDCA (20 µmol/l) for up to 60 min. Liver samples were taken at the time points indicated. The integrin antagonistic peptide (G RGD SP, 10 µmol/l), the inactive control peptide (G RAD SP, 10 µmol/l), the PI3-K inhibitor wortmannin (100 nmol/l), and the Src inhibitor PP-2 (250 nmol/l) were added 30 min prior to the addition of nor UDCA. Activation of Erk-1/-2, p38 MAPK and c-Src was analyzed by ( a,b ) Western blot using specific antibodies and ( c,d ) subsequent densitometric analysis. Total Erk-1/-2, total p38 MAPK , and total c-Src served as respective loading control. Phosphorylation at t = 0 min was arbitrarily set as 1. Densitometric analyses (means ± SEM) and representative blots of at least three independent perfusion experiments are shown. * p < 0.05 statistical significance compared with the unstimulated control. # p < 0.05 statistical significance between nor UDCA in the absence and presence of an inhibitor. nor UDCA led to a significant activation of Erk-1/-2, p38 MAPK as well as c-Src in the perfused rat liver, which was inhibited by G RGD SP, whereas G RAD SP had no effect. Phosphorylation of Erk-1/-2, p38 MAPK , and c-Src was sensitive to PP-2, whereas wortmannin inhibited Erk-1/2 and c-Src activation. Blots were cropped to focus on the area of interest, and full-length blots are presented in Supplementary Figs. and .

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Evidence for functional selectivity in TUDC- and nor UDCA-induced signal transduction via α 5 β 1 integrin towards choleresis

doi: 10.1038/s41598-020-62326-y

Figure Lengend Snippet: Model of α 5 β 1 integrin activation-dependent differential bile acid signaling. Activation of α 5 β 1 integrin with the less efficacious nor UDCA results in the formation of FAK Y397-P , which leads to c-Src- and PI3-K-dependent Erk-1/2 activation. When α 5 β 1 integrin is activated by the more efficacious TUDC, higher levels of FAK Y397-P result, which, in addition, trigger a slower activation of Erk-1/-2 via PI3-K in a c-Src-independent manner .

Article Snippet: The antibodies raised against the α 5 β 1 integrin dimer (AB1950) and the β 1 integrin subunit active conformation (#MAB2079Z), phospho-Erk-1/-2 (#9106), phospho-p38 MAPK (#9211), p38 MAPK (#9228), phospho-EGFR Tyr 1045 (#2237), phospho-Src-Tyr 418 (#2101), phospho-FAK Tyr 925 (#3284), and phospho-FAK Tyr 576/577 (#3281) were from Cell Signaling Technology, Inc. (Danvers, USA), against Erk-1/-2 (#06–182), EGFR (#06–847, Western blot, WB), Na + /K + -ATPase (#05–369), Cy3-conjugated donkey anti-rabbit IgG (#AP182C), and FITC-conjugated donkey anti-mouse IgG (#AP192C) from Merck-Millipore (Darmstadt, Germany).

Techniques: Activation Assay

Journal: Molecular Pharmaceutics

Article Title: Oral Administration and Detection of a Near-Infrared Molecular Imaging Agent in an Orthotopic Mouse Model for Breast Cancer Screening

doi: 10.1021/acs.molpharmaceut.7b00994

Figure Lengend Snippet: Macroscopic images of a tumor histology slide taken on an Odyssey CLx (agent) and Olympus FV1200 confocal microscope (other channels). The orally delivered agent shows a diffuse pattern in tumors compared to the negative control ( Figure S6 ). The intensity appears to be slightly higher in regions with higher macrophage density versus tumor cells or blood vessels (CD31). The slides were labeled with the AF680 agent ex vivo (integrin) and stained with Hoechst 33342 to show the presence of cells and integrin throughout the tissue. Scale bar = 500 μm.

Article Snippet: HEK-293 cells, which express endogenous α v but not β 3 were transfected with the β 3 integrin subunit (Addgene plasmid 27289) to generate an α v β 3 positive line.

Techniques: Microscopy, Negative Control, Labeling, Ex Vivo, Staining

Journal: Cell Death and Differentiation

Article Title: A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation

doi: 10.1038/cdd.2017.153

Figure Lengend Snippet: VnP-16 promotes osteogenic cell attachment through direct interaction with β1 integrin. (a) Attachment of human osteogenic cells pretreated with EDTA (5 mM), MnCl2 (500 μM), or heparin (100 μg/ml) to VnP-16. The cells were seeded onto plates that were precoated with VnP-16 (9.1 μg/cm2) for 1 h. (b) The effects of various integrin-blocking antibodies on cell attachment to VnP-16. (c–e) Immunoblotting (c) and densitometric analysis (d) of β1 integrin, and cell attachment to VnP-16 (e) in osteogenic cells that were transfected with a control (Con) or β1 integrin-specific siRNA (10 nM; β1 integrin). (f) Streptavidin-bead pulldown assay with the biotinylated SP or biotinylated VnP-16 peptides from extracts of osteogenic cells that were cultured on biotinylated SP- or biotinylated VnP-16-coated dishes for 30 min. Data in (a,b,e) (n=4), and (d) (n=2) represent the mean±SD. **P<0.01

Article Snippet: The cells (5 × 10 4 cells/250 μ l) were preincubated with 5 mM EDTA, 500 μ M MnCl 2 , 100 μ g/ml heparin, and 10 μ g/ml function-blocking antibodies against the integrin subunits α 1 (FB12), α 2 (P1E6), α 3 (P1B5), α 4 (P4C2), α 5 (P1D6), α 6 (NKI-GoH3), β 3 (B3A; Chemicon, Temecula, CA, USA), α v (AV1), or β 1 (6S6; Millipore) for 15 min at 37 °C.

Techniques: Cell Attachment Assay, Blocking Assay, Western Blot, Transfection, Control, Cell Culture

Journal: Cell Death and Differentiation

Article Title: A vitronectin-derived peptide reverses ovariectomy-induced bone loss via regulation of osteoblast and osteoclast differentiation

doi: 10.1038/cdd.2017.153

Figure Lengend Snippet: VnP-16 promotes osteogenic differentiation through β1 integrin/FAK signaling. (a,b) Immunoblotting (a) and densitometric analyses (b) of phospho-FAK, phospho-Akt Ser473, phospho-PKCδ Thr505, and phospho-c-Src Tyr416 in osteogenic cells that were cultured for 3 h on plates coated with vitronectin (0.23 μg/cm2), SP, or VnP-16 (9.1 μg/cm2). (c,d) Immunoblotting (c) and densitometric analyses (d) of total FAK (t-FAK) and phospho-FAK Tyr397 in osteogenic cells that were pretreated with PF-573228 for 1 h. (e) Attachment of cells that were treated with PF-573228 for 1 h in serum-free medium to plates precoated with VnP-16 (9.1 μg/cm2). (f) The effects of VnP-16 on alkaline phosphatase activity and calcium deposition in SKP-derived mesenchymal precursors (MPs), mouse calvarial osteoblast precursors (MC3T3-E1), and human osteogenic cells (Osteogenic). The cells were cultured in osteogenic differentiation medium containing VnP-16 or SP (50 μg/0.5 ml) for 2 weeks. (g) The effects of PF-573228 on alkaline phosphatase activity and calcium deposition in SKP-derived mesenchymal precursors, MC3T3-E1, and human osteogenic cells. The cells were cultured on VnP-16-treated (9.1 μg/cm2) plates in osteogenic differentiation medium with or without 1 μM PF-573228 for 2 weeks. (h–j) Immunoblotting (h) and densitometric analyses (i) of t-FAK, and dose-dependent attachment (j) of control or FAK-specific siRNA-treated (100 nM) osteogenic cells to VnP-16. (k) Determination of apoptotic cells in osteogenic cells that were cultured for 24, 48, and 96 h on plates coated with SP or VnP-16 (9.1 μg/cm2) by TUNEL assay. Data in (b, d and i) (n=3), and (e, j and k) (n=4) represent the mean±SD. *P<0.05 or **P<0.01 compared to vehicle or control siRNA

Article Snippet: The cells (5 × 10 4 cells/250 μ l) were preincubated with 5 mM EDTA, 500 μ M MnCl 2 , 100 μ g/ml heparin, and 10 μ g/ml function-blocking antibodies against the integrin subunits α 1 (FB12), α 2 (P1E6), α 3 (P1B5), α 4 (P4C2), α 5 (P1D6), α 6 (NKI-GoH3), β 3 (B3A; Chemicon, Temecula, CA, USA), α v (AV1), or β 1 (6S6; Millipore) for 15 min at 37 °C.

Techniques: Western Blot, Cell Culture, Activity Assay, Derivative Assay, Control, TUNEL Assay

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Storage modulus (Pa) of BME and soft and stiffPEG-HEP matrices (crosslinking degree of γ=0.63 and γ=1.25, respectively). (B) Top row: Bright field images of MEC colony morphology after culture in reference BME and soft and stiff PEG-HEP matrices (scale bar = 100 μm). Middle/bottom row: Confocal immunofluorescence of polarization markers in MEC colony cross sections from BME and soft and stiff PEG-HEP matrices (scale bar = 20 μm). Middle row: Immunofluorescence images of MECs stained for polarity markers β4-integrin (arrows; red) and β-catenin (arrowhead; green), and nuclei (DAPI; blue). Bottom row: Immunofluorescence images of MECs stained for laminin-332 (arrow; green), actin filaments (red), and nuclei (DAPI; blue). (C) Left: Percentage of acini containing cleared lumens. Right: Representative images of DAPI-stained acini showing luminal clearance. (D) Left: Percentage of acini containing apically oriented Golgi apparatus. Right: Representative images of GM-130- (Golgi apparatus) and DAPI-stained (nucleus) acini showing orientation of the Golgi relative to the nuclei. (E) Percentage of acini showing an invasive (non-spherical) phenotype. (F and G) Ultra-structural analysis of MECs cultured in soft PEG-HEP matrices. (F) Left image shows several desmosomes (red arrows) at the epithelial cell-cell interface (scale bar = 500 nm). Right image is a magnified inset of the left image showing the desmosomes (red arrows) with connected cytoplasmic filaments (red arrowheads, scale bar = 500 nm). (G) The top left overview image is a representative MEC colony with cleared lumen (L). Note that the hydrogel (G) polymeric network appears degraded in the periphery of the basal (B) acini surface (scale bar = 20 μm). The left bottom image is a magnified inset of the left top image (scale bar = 2 μm). The right image is further magnified at the cell-matrix interface, showing the typical dense dark structures of hemidesmosomes (arrows), which are connected to cytoplasmic filaments (arrowheads, scale bar = 500 nm). G = gel, L = lumen, B = basal side of acini. A, C, D and E are mean ± S.D. (A) ANOVA with Tukey's multiple comparison, n = 3. (C) and (D) n = 50 acini counted from three independent experiments; un-paired t-test. (E) n = 100-150 from one representative experiment; Kruskal-Wallis test with Dunn's multiple comparisons. All images and measurements in this figure are of cells after 14 days in culture. n.s. = not significant; p ≥ 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence, Staining, Cell Culture

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Storage modulus (Pa) of matrices with decreasing biomolecular polymer network functionalization. Left to right: MMP-cleavable PEG-HEP, MMP-insensitive PEG-scr-HEP, and MMP-cleavable PEG-PEG matrices. (B) Bright field images showing the colony morphology of MECs grown in soft synthetic matrices with different degrees of biofunctionality: (I) PEG-HEP, (II) PEG-scr-HEP, and (III) PEG-PEG matrices (scale bar = 200 μm). Bottom row: magnified image of the white box in top row (scale bar = 50 μm). (C) Confocal immunofluorescence images of MEC colony cross sections grown in the different matrices (as in B, scale bar = 20 μm). Top row: Immunfluorescence of β4-integrin (arrows; red), β-catenin (arrowhead; green), and nuclei (DAPI; blue). Bottom row: Immunfluorescence of the basement membrane protein laminin-332 (arrows; green), actin filaments (red), and nuclei (blue) (D) MECs were grown in PEG-MCP-HEP, PEG-scr-HEP, or PEG-MCP-PEG matrices, brightfield images were taken, and the colony diameters measured. (E) Percentage of acini containing cleared lumens. (F) Percentage of acini containing apically oriented Golgi apparatus. A, D, E and F are mean ± S.D. (A) ANOVA with Tukey's multiple comparison, n = 3; (E) and (F) unpaired t-test, n = 50 from three independent experiments. (D) Kruskal-Wallis test with Dunn's multiple comparisons; n= 100-150 from one representative experiment. All images and measurements in this figure are of cells after 14 days in culture. n.s. = not significant (p ≥ 0.05), ** p< 0.01, *** p< 0.001.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence

Journal: Biomaterials

Article Title: Modular GAG-matrices to promote mammary epithelial morphogenesis in vitro

doi: 10.1016/j.biomaterials.2016.10.007

Figure Lengend Snippet: (A) Top row: Confocal immunofluorescence images of laminin-332 (top, green) or laminin-111 (bottom, green) expression in MEC colonies grown in soft, degradable PEG-HEP hydrogels for the number of days indicated (scale bar = 20 μm). Top row: After two days laminin-332 was deposited partially around the outer colony surface. By day 4 of culture, laminin-332 appears fully distributed around the outer acini surface (actin filaments, red; nuclei, blue). Bottom row: No extracellular laminin-111 deposition was observed in the cultures. (B) MECs were grown in soft, degradable PEG-HEP matrices in the presence or absence of β1 or α6β4 integrin blocking antibodies. Brightfield images were taken, and the colony diameters measured. MEC colony growth, as measured by brightfield microscopy, is impaired by blocking β1 or α6β4 integrin function. (C) Colony diameter after 14 days. Kruskal-Wallis test with Dunn's multiple comparisons, n = 130-150 from one representative experiment. n.s. = not significant (P ≥ 0.05); *** P < 0.001. (D) Bright field images of MECs grown in soft, degradable PEG-HEP matrices after treatment with integrin blocking antibodies (β1 or α6β4; scale bar = 20 μm). (E) Diagram of the hypothesized mechanism of polarized acini development in soft, degradable PEG-HEP matrices. MECs embedded within soft, enzymatically degradable PEG-HEP hydrogels constitutively secrete LN-332, which binds to heparin and through binding to integrins acts as a signaling molecule to promote MEC morphogenesis into polarized acini.

Article Snippet: For functional integrin blocking studies antibodies directed against human integrin subunit β 1 (BD Biosciences 552828) and integrin heterodimer α 6 β 4 (α 6 (BD Biosciences 555734), β 4 (Millipore MAB2059)) were added to the PEG-MCP precursor solution and to the cell culture media at a final concentration of 20 μg/ml.

Techniques: Immunofluorescence, Expressing, Blocking Assay, Microscopy, Binding Assay