西亚试剂：Analysis of human CLCN4 SNVs in Xenopus oocytes

Analysis of human CLCN4 SNVs in Xenopus oocytes
CLCN4 SNVs were introduced into human CLCN4 (NM_001830.3; Gene ID: 118) cDNA cloned into pTLN and pCIneo36 by recombinant PCR. We assessed the expression level and stability of wild-type and mutants with p.Gly78Ser, p.Leu221Val, p.Val536Met or p.Gly731Arg substitutions by western blot analysis of lysates from transiently transfected cells using standard methods. Xenopus laevis oocytes were injected with 23 ng cRNA, which was transcribed with the mMessage Machine kit (Ambion, Thermo Fisher Scientific Inc., Waltham, MA, USA) from pTLN.37 After 3 days incubation at 17 °C, currents were measured at room temperature using standard two-electrode voltage clamp employing TurboTEC amplifiers (npi electronic, Tamm, Germany) and pClamp10.2 software (Molecular Devices, Sunnyvale, CA, USA). Oocytes were superfused with modified ND96 saline (96 mm NaCl, 2 mm K-gluconate, 1.8 mm Ca-gluconate, 1 mm Mg-gluconate, 5 mm HEPES pH 7.5) and clamped in 20-mV steps to voltages between −100 and +80 mV. The holding potential was −30 mV.

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