Journal: PLoS ONE

Article Title: Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

doi: 10.1371/journal.pone.0120886

Figure Lengend Snippet: A. Biosynthesis of constructs WT.AQP2 and CM.AQP2 following cotransin treatment. Columns represent the GFP fluorescence signals (arbitrary units) of the constructs after 19 h of treatment with 1.5% DMSO solvent alone (-) (dark grey columns) or 10 μM cotransin/1.5% DMSO (+) (light grey columns). Shown are mean values of three independent experiments ± SD. Fluorescence was quantified by flow cytometry measurements. B . Concentration-response curve for the cotransin-mediated biosynthesis inhibition of CM.AQP2. Data points represent the mean values of the GFP fluorescence signals of the construct after 19 h of treatment with increasing concentrations of cotransin (1–50 μM in 1.5% DMSO). Shown are mean values of three independent experiments ± SD. Fluorescence was quantified by flow cytometry measurements as above and data were normalized to the DMSO control (1.5% DMSO). The calculated IC 50 value is indicated. C . Upper Panel. Colocalization of the GFP fluorescence signals of the truncated constructs WT.AQP2.NT and CM.AQP2.NT (left side, green) with the CFP fluorescence signals of the cotransfected ER marker ECFP-ER (middle, red). The fluorescence signals were recorded using confocal LSM and computer-overlayed (right side; colocalization is indicated by yellow color). The xy-scans show representative cells and are representative of three independent experiments. The cartoon on the right side shows a schematic depiction of the constructs. The 4 point mutations of construct CM.AQP2.NT are indicated by (****). Lower panel. Subcellular localization of unfused soluble GFP (control protein which does not contain a signal sequence). For clarity, the GFP fluorescence signals (left side, green) and those of the plasma membrane dye trypan blue (middle, red) were recorded in this case and computer overlayed (right panel). The xy-scans show representative cells and are representative of three independent experiments.

Article Snippet: The plasma membrane stain trypan blue and the transfection reagent polyethylenimine (PEI) were from Merck-Millipore (Darmstadt, Germany).

Techniques: Construct, Fluorescence, Solvent, Flow Cytometry, Concentration Assay, Inhibition, Control, Marker, Sequencing, Clinical Proteomics, Membrane