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

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: British Journal of Cancer

Article Title: Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity

doi: 10.1038/sj.bjc.6605539

Figure Lengend Snippet: Suppression of cell–matrix adhesion and enhancement of the inhibitory effects of an integrin- β 1-function-blocking antibody by EG3287. ( A , C ) A549 and ACHN cells were pretreated for 30 min with the indicated concentrations of EG3287 and seeded to wells coated with the indicated matrix proteins. After 90 min of incubation, attached cells were labelled with calcein-AM and measured using a fluorescence plate reader. * P <0.05; ** P <0.01 vs untreated control. ( B , D ) A549 and ACHN cells were pretreated for 30 min with an integrin- β 1-blocking antibody at the indicated concentrations in the absence or presence of 100 μ M EG3287, and cell adhesion to matrix proteins was measured. * P <0.05; ** P <0.01 for integrin- β 1 antibody alone vs integrin- β 1 antibody plus EG3287.

Article Snippet: A functional blocking antibody against the integrin β 1-subunit was from Millipore (Livingston, UK).

Techniques: Blocking Assay, Incubation, Fluorescence, Control