Tetracycline regulated expression of mBest1 in mammalian cells. Expression of mBest1 was carried out using a tetracycline regulated expression system (T-Rex?, Invitrogen) as described previously for mBest3 [12]. A stable line expressing mBest1-c-myc was generated in TRex-293 Sorafenib (express tetracycline repressor protein). Transfected and untransfected TRex-293 cells were seeded onto coverslips 24–48 h before recordings or immunocytochemistry. mBest1 channel expression was induced by tetracycline (1 μg/ml) addition to the media. In some instances mBest1, mBest2 or mBest3 were transiently transfected into HEK TsA201 cells for patch clamp analysis. mBest3 in pcDNA4/TO/c-myc-HIS vector was generated as described [12]. mBest2 in pCMV-Sport6 (IMAGE clone ID 4989959) was obtained from Invitrogen. Each 35 mm culture dish was transfected with 2 μg plasmid DNA using Polyfect transfection reagent (Qiagen).
Data analysis.Erev and pharmacological percentage block were determined by curve fitting of individual current traces using Clampfit (PClamp, version 9.2; Molecular Devices). All data were exported to Origin 7.5 (OriginLab) or Graphpad PRISM 3.0 for plotting and curve fitting. PRISM 3.0 was used to determine statistical significance between groups with one-way ANOVA followed by Dunnett’s Multiple Comparison test. p
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Such low level of genetic differentiation
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