Journal: eLife

Article Title: Optogenetic control of excitatory post-synaptic differentiation through neuroligin-1 tyrosine phosphorylation

doi: 10.7554/eLife.52027

Figure Lengend Snippet:

Article Snippet: After blocking with 5% non-fat dried milk in Tris-buffered saline Tween-20 (TBST; 28 mM Tris, 137 mM NaCl, 0.05% Tween-20, pH 7.4) for 45 min at room temperature, membranes were probed for 1 hr at room temperature or overnight at 4°C with mouse anti-phosphotyrosine (1:1000, Cell Signaling Technology 9411S), rabbit anti-Nlgn1 (1:1000, Synaptic systems 129013), rabbit anti-FGFR1 (1:1000, Cell Signaling Technology D8E4), or rat anti-HA (1:1000, Roche 3F10).

Techniques: Transfection, Construct, Cloning, Plasmid Preparation, Electroporation, shRNA, Recombinant, Sequencing, Immunoprecipitation, Software

Journal: PLoS ONE

Article Title: LTBP-2 Has a Single High-Affinity Binding Site for FGF-2 and Blocks FGF-2-Induced Cell Proliferation

doi: 10.1371/journal.pone.0135577

Figure Lengend Snippet: A . The effect of LTBP-2 on the bio-activity of FGF-2 was tested in a cell proliferation assay (see experimental). Human foreskin fibroblasts were treated with FGF-2 with and without follistatin (white columns), or FGF-2 and follistatin pre-incubated with 5 or 10 fold molar excess of full length LTBP-2 or fragment LTBP-2C F2 (cross-hatched). Negative controls (black columns), included cells only and cells incubated with follistatin, LTBP-2 or fragment LTBP-2C F2. Mean values ± S.D. from triplicate determinations. Note 5 fold molar excess of full-length LTBP-2 completely blocked FGF-2 induced cell proliferation (p = 0.0001) and 5-fold molar excess of fragment LTBP-2C F2 partially blocked the activity (p = 0.0001). B . Immunoblot analysis FGF receptor (FGFR1) phosphorylation. Human foreskin fibroblasts were treated for 2 hours with FGF-2 (10 ng / ml) only or with FGF-2 plus 10-fold molar excess of full length LTBP-2 (LTBP-2 FL) or fragment F2 (LTBP-2C F2). Control cells had no FGF-2 or LTBP-2 added. Cellular proteins were extracted and duplicate samples were analysed by SDS-PAGE and immunoblotting with anti-phospho-FGFR1 antibody, and anti-total FGFR1 antibody. Bands were visualised using the LI-COR Odyssey Infrared Imaging System. C . The band intensity was measured using ImageJ 1.48 software [National Institutes of Health (NIH), Bethesda, MD] and normalised to the internal β actin signal. The ratio of the phospho-FGFR1 to total FGFR1 value for each sample is expressed relative to the average FGF-2 only control value (= 100%). Note the strong FGFR1 activation by FGF-2 was substantially blocked by both LTBP-2 C and LTBP-2C F2 fragments. Mean values ± S.D. of duplicate lanes.

Article Snippet: Rabbit monoclonal anti-total FGFR1 antibody (D8E4) was purchased from Cell Signalling Technology (Danvers, MA).

Techniques: Activity Assay, Proliferation Assay, Incubation, Western Blot, Phospho-proteomics, Control, SDS Page, Imaging, Software, Activation Assay