rat anti human α6-integrin functional blocking antibody goh3  (Millipore)

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Structure of human tenascin-C. A, Organization of the tenascin-C molecule. This diagram is adapted from Aukhil et al. (1993). The N termini of three arms are joined to form a trimer, and two trimers are connected via a disulfide bond (S-S) to form a hexamer. Each arm consists of 14 domains with homology to epidermal growth factor (EGF), 8-17 FN-III repeats depending on alternative RNA splicing, and a single fibrinogen (fbg) domain. The universal FN-III repeats (fn1-5 and fn6-8) are present in all tenascin-C splice variants. The largest tenascin-C splice variant contains nine alternatively spliced FN-III repeats (designated A1, A2, A3, A4, B, AD1, AD2, C, and D, or fnA-D), which are missing in the shortest splice variant. B, Crystal structure of universal tenascin-C FN-III domain 3 (fn3). This structure is adapted from Leahy et al. (1992) and consists of six exposed loops and seven β strands. The α9β1 integrin recognition sequence EIDGIELT, which corresponds to the neurite outgrowth-promoting sequence VFDNFVLK in fnD, is highlighted and includes portions of an exposed loop and adjacent β strand.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Variant Assay, Sequencing

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: A β1 integrin neuronal receptor is implicated in promotion of neurite outgrowth by fnD and VFDNFVLK. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound fnD or VFDNFVLK and a function-blocking antibody againstβ1 integrin subunit (10 μg/ml). Distributions of total neurite length are presented as a box-and-whisker plot. One representative experiment of four is shown. Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers indicate 5th and 95th percentiles. FnD and VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody did not alter neurite outgrowth on PLL but completely blocked outgrowth promotion by fnD and VFDNFVLK. B, Neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound plus excess soluble (bd + sl) VFDVFVLK and β1 integrin antibody. One representative experiment of three is shown. Outgrowth values were higher than those observed in A for neurons cultured on bound peptide alone and reduced to control values in the presence of β1 antibody.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay, Whisker Assay, Control

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Neurons cultured on bound VFDNFVLK but not VSPNGSLK demonstrate clustering of immunoreactivity for β1 integrin subunit. Immunocytochemistry was performed using a monoclonal hamster antibody against β1 integrin chain, followed by a fluorescein-conjugated goat anti-hamster secondary antibody. VFDNFVLK but not VSPNGSLK induced clustering of neuronal β1 integrin immunoreactivity. Scale bar, 10 μm.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Immunocytochemistry

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Cerebellar granule neurons express the X2B isoform of the α7 integrin subunit. A, Homogenate from P8 rat cerebellar granule neurons (25 μg) was separated on 8% SDS-PAGE gels under nonreducing conditions and then transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody, which cross-reacts with all splice variants of the α7 integrin chain, or polyclonal α7A or α7B antibodies, which cross-react with variants containing the A or B cytoplasmic tail. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots probed with the O26 or α7B antibody demonstrated one predominant polypeptide band with Mr of 120,000, whereas blots probed with the α7A antibody revealed no polypeptide band. B, RT-PCR was performed on total RNA prepared from P8 rat cerebellar granule neurons. Amplification was done using sense and antisense primers designed to amplify X1, X2, A, and B variants of the α7 integrin as described in Materials and Methods. The 200 bp fragment corresponds to the X2 extracellular variant, and the 370 bp fragment corresponds to the B cytoplasmic variant. No products were obtained that corresponded to the X1 extracellular variant (220 bp) or the A cytoplasmic variant (480 bp).

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: SDS Page, Reverse Transcription Polymerase Chain Reaction, Amplification, Variant Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: The α7 integrin subunit is implicated as the partner for β1 in promotion of neurite outgrowth by VFDNFVLK. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated glass coverslips in the presence of bound VFDNFVLK and function-blocking O26 antibody against α7 integrin subunit (100, 50, 10, 5, 1, or 0.5 μg/ml). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with the PLL control (*p < 0.05; Kolmogorov-Smirnov test). The α7 integrin antibody did not alter neurite outgrowth on PLL at any concentration used. The antibody inhibited promotion of outgrowth by VFDNFVLK and demonstrated a biphasic concentration dependence. Outgrowth was significantly impaired at 50, 10, and 5 μg/ml O26 antibody (**p < 0.05; Kolmogorov-Smirnov test), with maximal inhibition at 10 μg/ml.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay, Control, Concentration Assay, Inhibition

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: The α1, α2, α3, and α6 integrin subunits do not play a role in VFDNFVLK-facilitated neurite growth. Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound VFDNFVLK and function-blocking antibodies against α1, α2, or α3 integrin subunits (10 μg/ml) (A), whereas mouse cerebellar granule neurons were cultured in the presence of bound VFDNFVLK and a function-blocking antibody against α6 integrin subunit (10 μg/ml) (B). One representative experiment of three is shown. VFDNFVLK significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). None of the antibodies altered neurite outgrowth on PLL or VFDNFVLK.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: α7 integrin is involved in neurite outgrowth promotion by tenascin-C and laminin-1 but not by fibronectin. A, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound large tenascin-C and function-blocking antibody O26 against α7 integrin subunit or function blocking antibody against β1 integrin subunit. One representative experiment of three is shown. Tenascin-C significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially blocked outgrowth promotion by large tenascin-C; the reduction in outgrowth values was significant (**p < 0.05; Kolmogorov-Smirnov test). The β1 integrin antibody completely blocked outgrowth promotion by large tenascin-C. B, Rat cerebellar granule neurons were cultured for 24 hr on PLL-coated coverslips in the presence of bound laminin-1 or fibronectin and function-blocking O26 antibody against α7 integrin subunit. One representative experiment of three is shown. Laminin-1 and fibronectin significantly enhanced outgrowth compared with PLL (*p < 0.05; Kolmogorov-Smirnov test). The α7 antibody partially reduced neurite outgrowth on laminin-1; the reduction was significant (**p < 0.05; Kolmogorov-Smirnov test). The antibody did not alter neurite outgrowth on fibronectin.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Cell Culture, Blocking Assay

Journal: The Journal of Neuroscience

Article Title: Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

doi: 10.1523/JNEUROSCI.4519-03.2004

Figure Lengend Snippet: Neuronal α7β1 integrin binds to D5 peptide in an FD-FV-dependent manner. P8 cerebellar granule neuronal extracts were incubated with Sepharose beads conjugated with either wild-type D5 peptide (D5 wt) or D5 mutant 1 peptide with alterations in both FD and FV (D5 mt). A, Proteins eluted with EDTA from wild-type or mutant D5 peptide were separated on 8% SDS-PAGE gels under nonreducing conditions and transferred to nitrocellulose paper. Blots were probed with monoclonal O26 antibody against α7 integrin chain or polyclonal antibody against β1 integrin chain. Immunoreactive bands were visualized using the enhanced chemiluminescence method. Blots of proteins eluted with EDTA from D5 wt peptide revealed polypeptide bands with Mr of 120,000 and 116,000, respectively, for the O26 and β1 integrin antibodies. Blots of proteins eluted with EDTA from D5 mt peptide revealed no polypeptide bands. B, Silver-stained gels revealed overlapping polypeptide bands with Mr of 120,000 and 116,000 for proteins eluted with EDTA from D5 wt peptide but not D5 mt 1 peptide.

Article Snippet: Function-blocking hamster monoclonal antibody against mouse α6 (GoH3) integrin chain was purchased from Immunotech (Marseille, France).

Techniques: Incubation, Mutagenesis, SDS Page, Staining

Journal:

Article Title: Division of Labor among the ?6?4 Integrin, ?1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and ?-Casein Expression in Mammary Epithelial Cells

doi:

Figure Lengend Snippet: Inhibition of β-casein expression by function-blocking integrin antibodies. Assays for the induction of β-casein expression in SCp2 mammary epithelial cells were performed on cells initially spread on plastic and subsequently exposed to medium containing the lactogenic hormone prolactin, plus or minus laminin, and function-blocking antibodies against the β1, α6, α1, α5, and αv integrin subunits. (A) Schematic representation of the assay. A two-step signaling process leads to β-casein expression after cell contact with laminin, beginning with a prerequisite cell shape change (rounding), followed by subsequent biochemical signaling events. (B) β-Casein expression was assayed by immunoblots of cell extracts and appears as a doublet migrating at ∼34 kDa. Laminin-induced β-casein expression was inhibited in the presence of β1 and α6 integrin-blocking antibodies. (C) Titration of the α6-blocking (GoH3) antibody shows maximal inhibition in the range of 2–5 μg/ml.

Article Snippet: The function-blocking integrin antibodies against α1 (Ha31/8), α5 (5H10-27), α6 (GoH3), αv (H9.2B8), and β1 (Ha2/5 and 9EG7) subunits were purchased as azide- and endotoxin-free reagents from PharMingen (San Diego, CA).

Techniques: Inhibition, Expressing, Blocking Assay, Western Blot, Titration

Journal:

Article Title: Division of Labor among the ?6?4 Integrin, ?1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and ?-Casein Expression in Mammary Epithelial Cells

doi:

Figure Lengend Snippet: Morphogenic changes induced by laminin are unaltered by the presence of α6- and β1-blocking antibodies. Assays for the induction of β-casein expression in SCp2 mammary epithelial cells were performed on cells initially spread on plastic and subsequently exposed to medium containing the lactogenic hormone prolactin, plus or minus laminin, and function-blocking antibodies against the β1 and α6 integrin subunits. In the absence of added laminin, cells remained attached and spread on the plastic and continued to grow to confluence (A). In contrast, cells exposed to laminin underwent cell rounding, and those in contact with other cells clustered into multicellular aggregates, leaving much of the plastic culture dish exposed (B). Cells exposed to laminin in the presence of function-blocking antibodies against the α6 (C) and β1 (D) integrin subunits continued to undergo the cell shape changes induced by laminin even though these same antibody treatments perturbed signals for β-casein expression.

Article Snippet: The function-blocking integrin antibodies against α1 (Ha31/8), α5 (5H10-27), α6 (GoH3), αv (H9.2B8), and β1 (Ha2/5 and 9EG7) subunits were purchased as azide- and endotoxin-free reagents from PharMingen (San Diego, CA).

Techniques: Blocking Assay, Expressing

Journal:

Article Title: Division of Labor among the ?6?4 Integrin, ?1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and ?-Casein Expression in Mammary Epithelial Cells

doi:

Figure Lengend Snippet: β-Casein expression and cell survival in the presence of function-blocking antibodies against β1 and α6 integrins in prerounded cells. SCp2 cells cultured in suspension were treated with prolactin, plus or minus laminin, and function-blocking antibodies against the β1, α6, α1, α5, and αv integrin subunits. (A) Assays of β-casein expression in suspension cultures measure signaling events subsequent to, and independent of, the required cell shape changes. (B) Immunoblots of cell extracts after 3 d of laminin exposure show that laminin-induced β-casein expression in prerounded cells was still inhibited in the presence of β1 and α6 integrin-blocking antibodies. (C) Cell viability was assayed by Alamar Blue dye reduction in duplicate wells under β-casein assay conditions identical to those described for B, but culture was for 4 d (1 d longer) to measure any cell death that may have been initiated during the course of the β-casein assay.

Article Snippet: The function-blocking integrin antibodies against α1 (Ha31/8), α5 (5H10-27), α6 (GoH3), αv (H9.2B8), and β1 (Ha2/5 and 9EG7) subunits were purchased as azide- and endotoxin-free reagents from PharMingen (San Diego, CA).

Techniques: Expressing, Blocking Assay, Cell Culture, Western Blot

Journal:

Article Title: Division of Labor among the ?6?4 Integrin, ?1 Integrins, and an E3 Laminin Receptor to Signal Morphogenesis and ?-Casein Expression in Mammary Epithelial Cells

doi:

Figure Lengend Snippet: Assays of β-casein expression, in the absence of added laminin, in primary cell cultures, clonal epithelial cells, and cocultures of clonal epithelial cells and fibroblasts. (A) Primary murine mammary epithelial cell cultures, SCp2 clonal epithelial cells, and cocultures of SCp2 cells and NIH3T3 fibroblasts (10:1) were assayed for β-casein expression in both flat cells (F) and rounded cells (R) (suspension culture) exposed to prolactin in the absence of added laminin. Cell rounding in suspension cultures permitted the induction of β-casein in primary cultures but not in the clonal SCp2 cell line. Coculture of SCp2 cells with a mesenchymal component (NIH3T3 fibroblasts) permitted the induction of β-casein. The same immunoblot filters were also probed for E-cadherin to demonstrate normalization for equal cell number. (B) SCp2 cells and NIH3T3 fibroblasts were cocultured in suspension (prerounded) and exposed to prolactin in the presence of function-blocking antibodies against the β1, α6, α1, α5, and αv integrin subunits, without the addition of laminin. β-Casein expression induced by endogenous basement membrane formation was inhibited by the β1 integrin-blocking antibody but was not inhibited effectively by the α6 integrin-blocking antibody GoH3.

Article Snippet: The function-blocking integrin antibodies against α1 (Ha31/8), α5 (5H10-27), α6 (GoH3), αv (H9.2B8), and β1 (Ha2/5 and 9EG7) subunits were purchased as azide- and endotoxin-free reagents from PharMingen (San Diego, CA).

Techniques: Expressing, Western Blot, Blocking Assay