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Quest C1 Esterase Inhibitor

PCR products were generated with unique restriction sites on their ends, and the fragments to be subcloned were directionally ligated into the polylinker of the appropriate pGEX vector. All PCR products were verified by DNA sequencing and found to be free of mutations.Blots were developed using enhanced chemiluminescence. Peptides were coupled to an aminoterminal biotin via the fourresidue spacer SGSG and purified by high pressure liquid chromatography.Sections from human bronchi were prepared as described.Samples were washed in TEE and bound proteins removed from the beads by boiling in sample buffer.In some experiments, unbound fractions were precipitated by addition of ml of acetone overnight at C.Although CFTR was not stable when precipitated in acetone overnight, we observed CFTR proteolytic fragments with antisera to the R domain or COOH terminus. Bound and unbound fractions were separated on SDSPAGE gels and analyzed by immunoblotting with EBP or CFTR antisera.Biotinylated peptides were immobilized onto neutravidincoated CM sensor chips. Purified GSTEBP fusion proteins were injected onto the Beclomethasone dipropionate peptide surfaces at a flow rate of mlmin.Immunohistochemical analysis of sections from human bronchi using antisera directed against EBP demonstrated intense staining of the apical cell surface with little staining of cilia or internal structures. Both ezrin and EBP are Beta-Cyclodextrin expressed in several epithelial cell lines derived from bronchus, colon, and kidney. Multiple EBP species are seen in all the cell lines shown, most likely representing differentially phosphorylated forms, so these proteins could form part of a subapical membraneanchoring complex specific to epithelia.CalU cells are a human airway epithelial cell line that expresses robust levels of CFTR.CFTR was present only in the particulate fraction, whereas EBP and ezrin were distributed equally in soluble and particulate fractions. Proteins were visualized following incubation with horseradish peroxidaseconjugated secondary antisera and ECL reagent.Endogenous fulllength CFTR was recovered bound to the GSTEBP beads but not to GST beads, indicating that EBP and CFTR are capable of interacting.These results suggest that the COOH terminus of CFTR is involved in the interaction with EBP.Because the CFTR peptide, in which the final four amino acid residues were replaced by glycines, the last four residues of CFTR must play a critical role in the association.These data demonstrate that EBP and CFTR interact and further demonstrates that the COOH terminus of CFTR is sufficient to mediate the interaction.To test the specificity of the interaction between CFTR and EBP, we performed assays using a peptide derived from the COOH terminus of the skeletal muscle voltagegated sodium channel as an affinity ligand.The reciprocal affinity purifications demonstrate that EBP and CFTR proteins associate and that the association is mediated by the COOH terminus of CFTR.The bound fraction was electrophoresed on SDSPAGE and immuoblotted with rabbit antiCFTR R domain. We used a combination of in vitro binding assays, gel overlays, and surface plasmon resonance to determine whether CFTR and EBP associate directly.EBP contains two PDZ domains and has an ezrin binding site at its extreme COOH terminus. We also demonstrated a direct interaction between the COOH terminus of CFTR and fulllength EBP in gel overlay assays.

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