The antisense and sense probes span their entire coding sequences of eGFP and RFP and were synthesized from linearized pCS2-eGFP and pCAGGS-RFP plasmids

a CD40 results in transient NFkB activation whereas AP-1 and STAT 3 activation 15272207 is upregulated and sustained. In view of these findings, and the antiapoptotic effect of C4BP we investigated NFkB cFos/cJun and STAT 3 activation following C4BP/sCD154 co-stimulation. Western blotting with specific antibodies to the functional transcription factors was carried out on cellular nuclear protein extracts. This approach has been previously described and shown to agree with results obtained by Electrophoretic Mobility Gel Shift Assay. Cholangiocyte monolayers were treated with sCD154, C4BP or both for either 4 or 24 hours. After these times, monolayers were harvested by scraping into cold PBS. Nuclear protein extracts were prepared as previously described and protein content of each sample determined using the Micro Lowry Total Protein Kit. Samples were resolved on a 10% Bis-Acrylamide gel by SDS-PAGE, followed by transfer to nitrocellulose membrane. Membranes were blocked overnight at 4uC in phosphate-buffered saline containing 5% w/v non-fat dried milk and then AZ-505 web washed with 0.1%Tween-20/PBS, before incubating with primary antibodies. All incubations were for 1 hour at room temperature in Tween/PBS containing 5% w/v non-fat dried milk. The following antibodies were used; a) NFkB mouse monoclonal primary antibody at 1/3000 dilution. b) c-Fos rabbit polyclonal primary antibody at 1/2000 dilution. c) c-Jun rabbit polyclonal primary antibody at 1/2000 dilution. d) STAT3 mAb at 1/1000 dilution. e) phospho-STAT3 mAb at 1/1000 dilution, UMR 538, CHU Saint Antoine, Paris, France, 2 Universite Pierre et Marie Curie, 1 Institut National de la Sante et de la Recherche Me CHU Saint Antoine, Paris, France, 3 UMR Centre National de la Recherche Scientifique 7613, Universite Pierre et Marie Curie, Paris, France Background. Basic cell-penetrating peptides are potential vectors for therapeutic molecules and display antimicrobial activity. The peptide-membrane contact is the first step of the sequential processes leading to peptide internalization and cell activity. However, the molecular mechanisms involved in peptide-membrane interaction are not well understood 14642775 and are frequently controversial. Herein, we compared the membrane activities of six basic peptides with different size, charge density and amphipaticity: Two cell-penetrating peptides, three amphipathic peptides and the neuromodulator substance P. Methodology/Principal Findings. Experiments of X ray diffraction, video-microscopy of giant vesicles, fluorescence spectroscopy, turbidimetry and calcein leakage from large vesicles are reported. Permeability and toxicity experiments were performed on cultured cells. The peptides showed differences in bilayer thickness perturbations, vesicles aggregation and local bending properties which form lipidic tubular structures. These structures invade the vesicle lumen in the absence of exogenous energy. Conclusions/Significance. We showed that the degree of membrane permeabilization with amphipathic peptides is dependent on both peptide size and hydrophobic nature of the residues. We propose a model for peptide-induced membrane perturbations that explains the differences in peptide membrane activities and suggests the existence of a facilitated physical endocytosis,which represents a new pathway for peptide cellular internalization. `re Citation: Lamazie A, Burlina F, Wolf C, Chassaing G, Trugnan G, et al Non-Metabolic Membrane Tubulation and Permeability Induced by B

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