Journal: Nature cell biology

Article Title: Integrin endosomal signalling suppresses anoikis

doi: 10.1038/ncb3250

Figure Lengend Snippet: a, Representative immunoblot of subcellular fractionation of FAK+/+ and FAK-/- MEFs (five independent experiments). b, Representative immunoblot of recombinant FAK (phosphorylated/activated or non-activated: n.a) recruitment to insoluble endosomal pellet (P) and soluble supernatant (S) fractions isolated from FAK -/- MEFs (five independent experiments). c, Representative immunoblot analysing the recruitment of recombinant FAK to purified endosomes derived from either control- or β1-integrin-silenced FAK -/- MEFs (five independent experiments). d, Representative immunoblot analysing the activation of recombinant FAK in purified endosome fractions derived from FAK -/- MEFs in the presence or absence of 10 µM ATP (five independent experiments). e, Subcellular fractionation of FAK+/+ MEFs transfected with GFP-FAK FAT or GFP-FAK FERM. Shown are representative immunoblots of GFP-FAK fragments and endogenous FAK (total-FAK) from two independent experiments. f, MDA-MB-231 cells plated on 20 μm round fibronectin-coated micropatterns (45 min) ± dynasore. Representative maximum projections and quantification of pFAK (mean fluorescence ± SEM, n=3 independent experiments, 10 cells analysed / experiment). Lys: cell lysate; PM: plasma membrane; Cyto: cytosol; Endo: Endosomal fraction. Student's two-tailed unpaired t-test P =0.01. Uncropped images of blots are shown in supplementary figure 9.

Article Snippet: Therefore, the endosomal fraction derived from FAK -/- MEFs was resuspended in buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 1 mM DTT and protease & phosphatase inhibitors and subsequently incubated with 0.5 μg of either phosphorylated or non-phosphorylated FAK (ProQinase) or GST for 2 h at room temperature.

Techniques: Western Blot, Fractionation, Recombinant, Isolation, Purification, Derivative Assay, Control, Activation Assay, Transfection, Fluorescence, Clinical Proteomics, Membrane, Two Tailed Test

Journal:

Article Title: New functional roles for non-collagenous domains of basement membrane collagens

doi:

Figure Lengend Snippet: Functions of NC1 and NC1 fragments of collagen IV, XV and XVIII

Article Snippet: , FAK and PI3K phosphorylation (α3) ( Pasco et al., 2000a ) , .

Techniques: Inhibition, Chemotaxis Assay, Phospho-proteomics, Activation Assay, Migration, In Vitro, Membrane, Angiogenesis Assay

Journal:

Article Title: New functional roles for non-collagenous domains of basement membrane collagens

doi:

Figure Lengend Snippet: Biological activities of immobilized versus soluble NC1 and endostatin fragments. Immobilized NC1 domains from collagen IV, XV and XVIII induce proliferation, survival and migration of different cell types. These effects correlate with an increase in PI3K, FAK and paxillin phosphorylation. By contrast, soluble NC1 or endostatin fragments bind to various receptors on the surface of the cells and decrease phosphorylation of PI3K, FAK and paxillin. Endostatin as well as NC1(IV) induces a decrease in the transcription of different genes. Endostatin interacts with VEGF-R2 and thus decreases the binding of VEGF to its receptor; endostatin also interacts directly with MMP-2, inhibiting the activation of the enzyme. Taken together, these soluble fragments act in the opposite way to their original molecules, negatively regulating the proliferation and the migration of different cell types and inducing apoptosis and extracellular matrix disorganization.

Article Snippet: , FAK and PI3K phosphorylation (α3) ( Pasco et al., 2000a ) , .

Techniques: Migration, Phospho-proteomics, Binding Assay, Activation Assay