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, Daejeon, Korea) under the following amplification conditions: 94?��C for 3?min followed by 50 cycles of 94?��C for 1?min, 62?��C for 10?s and 72?��C for 15?s, and final extension was at 72?��C for 15?min. The PCR products were then purified using a QIAquick Gel Extraction Kit (Qiagen, D��sseldorf, Germany) and directly sequenced using a cycle method with the same primers for PCR and a Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) with the following conditions: 96?��C for 5?min followed by 24 cycles of 96?��C for 10?s, 50?��C for 5?s and 60?��C for 4?min and Ritipenem final extension at 72?��C for 5?min, in conjunction with an ABI Prism 3500dx automated genetic analyzer (Applied Biosystems). For single-strand DNA PCR, the GJB2 gene was amplified with the primers forward 5��-ATGGATTGGGGCACGCTGC-3�� Volasertib datasheet and reverse 5��-ACGTACATGAAGGCGGCTTCG-3��. The 458-bp PCR product was inserted into the T-blunt vector (SolGent, Daejeon, Korea). After transformation, a single colony was selected, and conventional nucleotide sequencing was performed. Molecular cloning and transfection of HEK293T cells HEK-293T cells obtained from the Korean Cell Line Bank (Seoul, Korea) were maintained in Dulbecco's modified Eagle's medium-HG (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and penicillin (50?IU?ml-1)/streptomycin (50?��g?ml-1). The coding region of human Cx26 complementary DNA was subcloned into the pEGFP-N1 mammalian expressible plasmid using BamHI and EcoRI restriction sites. Plasmids expressing Cx26-V37I and Cx26-R75Q mutant proteins were generated using a PCR-based site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The following primers were used for Cx26-V37I and Cx26-R75Q mutagenesis, respectively: p.V37I (forward) 5��-TATGATCCTCATTGTGGCTGCAAAGGAGGTG-3��, pV.37I (reverse) 5��-GCAGCCACAATGAGGATCATAATGCGAAAAATG-3��, p.R75Q (forward) 5��-CCACATCCAGCTATGGGCCCTGCAGCT-3��, p.R75Q (reverse) 5��-CCATAGCTGGATGTGGGAGATGGGGAAG-3��. Plasmids were transiently transfected into HEK-293T cells using Lipofectamine Plus Reagent (Invitrogen). Biochemical coupling assay and hemichannel permeability assay Gap junction-mediated biochemical coupling and hemichannel (connexon) permeability CB-839 nmr was measured as described previously.13 Computational modeling SWISS-MODEL was used to generate model of GJB2 tertiary structure as previously reported.14 Cx26-R75Q and Cx26-V37I+R75Q modelings were based on PDB file 2ZW3 template15. Molecular graphics and analyses were performed with the USCF Chimera package (http://www.cgl.ucsf.edu/chimera). Statistical analysis The results of multiple experiments are presented as mean��s.e.m. Statistical analysis was performed using Student's t-tests or analysis of variance followed by Tukey's multiple comparison test as appropriate. P
