fak inhibitor 14 (faki, sml0837)  (Millipore)

Journal: PLoS ONE

Article Title: CCN1 Induces Oncostatin M Production in Osteoblasts via Integrin-Dependent Signal Pathways

doi: 10.1371/journal.pone.0106632

Figure Lengend Snippet: (A) Osteoblasts were pretreated with FAK inhibitor (FAKi) (10 µM) or transfected with FAK siRNA (2 µm) for 24 h, then stimulated by CCN1, OSM expression gauged by qPCR. (B) Cells were transfected with FAK siRNA (2 µm) or pretreated with FAKi, protein level of FAK measured by western blot (upper-panel), OSM expression rated by ELISA assay (lower-panel). (C) Cells pretreated with FAKi for 30 min were stimulated with CCN1, protein level of OSM was measured by western blot. (D) Osteoblasts were incubated with CCN1 in time intervals, and p-FAK expression derived by western blot. (E&F) Cells pretreated with αvβ3, αvβ5, α5β1, and α6β1 antibody for 30 min were stimulated with CCN1, p-FAK expression rated by western blot. Data represent mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCN1-treated group.

Article Snippet: Human recombinant CCN1 was obtained from PeproTech (Rocky Hill, NJ), FAK inhibitor (FAKi) and c-Src inhibitor (PP2), PI3K inhibitors (Wortmannin and Ly294002), NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK) from Sigma-Aldrich (St. Louis, MO).

Techniques: Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Derivative Assay

Journal: PLoS ONE

Article Title: CCN1 Induces Oncostatin M Production in Osteoblasts via Integrin-Dependent Signal Pathways

doi: 10.1371/journal.pone.0106632

Figure Lengend Snippet: (A) Osteoblasts were pretreated with c-Src inhibitor, PP2, or transfected with FAK siRNA followed by stimulation with CCN1, OSM expression measured by qPCR. (B) Cells were transfected with c-Src siRNA (2 µm) or pretreated with PP2, protein level assessed by western blot (upper-panel), OSM expression measured by ELISA assay (lower-panel). (C) Cells pretreated with PP2 for 30 min follow were stimulated with CCN1, protein level of OSM measured by western blot. (D) Cells were incubated with CCN1 in time intervals, p-c-Src expression rated by western blot. (E) Cells pretreated with FAKi for 30 min were stimulated by CCN1, p-c-Src expression rated by western blot. (F&G) Cells pretreated with αvβ3, αvβ5, α5β1, and α6β1 antibody for 30 min were stimulated with CCN1, p-c-Src expression rated by western blot. Data represent mean ± S.E. *, p<0.05 compared to control; #, p<0.05 compared with CCN1-treated group.

Article Snippet: Human recombinant CCN1 was obtained from PeproTech (Rocky Hill, NJ), FAK inhibitor (FAKi) and c-Src inhibitor (PP2), PI3K inhibitors (Wortmannin and Ly294002), NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK) from Sigma-Aldrich (St. Louis, MO).

Techniques: Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation

Journal: PLoS ONE

Article Title: CCN1 Induces Oncostatin M Production in Osteoblasts via Integrin-Dependent Signal Pathways

doi: 10.1371/journal.pone.0106632

Figure Lengend Snippet: (A) Osteoblasts were incubated with various concentrations of CCN1 (3–30 ng/ml) in NF-κB luciferase activity. (B&C) Osteoblasts pretreated with FAK inhibitor, PP2, Wortmannin, Ly294002, PDTC, and TPCK for 30 min or transfected with FAK, c-Src, PI3K, and p65 siRNA were treated with CCN1. NF-κB luciferase activity measured, results normalized to β-galactosidase activity and expressed for three independent experiments performed in triplicate. (D) Osteoblasts pretreated with FAKi, PP2, Wortmannin, LY294002 for 30 min were stimulated with CCN1 for 60 min, followed by chromatin immunoprecipitation assay. (E) Schematic diagram of signal pathway showed CCN1-induced OSM expression in osteoblasts. Data represent mean ± S.E. *, p<0.05 compared with control; #, p<0.05 compared with CCN1-treated group.

Article Snippet: Human recombinant CCN1 was obtained from PeproTech (Rocky Hill, NJ), FAK inhibitor (FAKi) and c-Src inhibitor (PP2), PI3K inhibitors (Wortmannin and Ly294002), NF-κB inhibitors pyrrolidine dithiocarbamate (PDTC) and L-1-tosylamido-2-phenylenylethyl chloromethyl ketone (TPCK) from Sigma-Aldrich (St. Louis, MO).

Techniques: Incubation, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, Expressing