Expression and Characterization of KCNG4 Mutant

A full-length human KCNG4 cDNA clone was purchased from Source bioscience (IRCMp5012B0629D) and cloned in-house into a pcDNA3 based expression plasmid (CMV-genex-polioIRESmCherry) both with or without a C-terminal HA tag. The p.Val419Met mutation was introduced by site-directed mutagenesis (Stratagene) according to the manufacturer’s protocols and sequences of the plasmids were confirmed by Sanger Sequencing. The clone expressing K_V2.1 alongside a nuclear GFP reporter in the pCAGGS-IRES2-nucEGFP vector has been described previously (Saitsu et al., 2015 (link)) and was a kind gift from Prof. Hiromoto Saitsu.
Antibodies used were anti-HA mouse monoclonal (12B12, #MMS-101P, Biolegend), anti-Na⁺/K⁺ ATPase rabbit monoclonal (ab76020, Abcam), anti-mCherry rat monoclonal (M11217), anti-β-actin mouse monoclonal (a2228, Sigma), anti-K_V2.1 rabbit polyclonal (APC-012, Alomone), anti-K_V6.4 mouse monoclonal (N458/10, NeuroMab), anti-K_V2.1 mouse monoclonal (K89/34, ab192761, Abcam), and anti-HA rabbit monoclonal (C29F4 #3724, Cell Signaling).

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Lee M.C., Nahorski M.S., Hockley J.R., Lu V.B., Ison G., Pattison L.A., Callejo G., Stouffer K., Fletcher E., Brown C., Drissi I., Wheeler D., Ernfors P., Menon D., Reimann F., Smith E.S, & Woods C.G. (2020). Human Labor Pain Is Influenced by the Voltage-Gated Potassium Channel KV6.4 Subunit. Cell Reports, 32(3), 107941.

Publication 2020

, #MMS-101P ab76020 M11217 a2228 APC-012 ab192761

Actin Antibodies Cdna Clone Kcng4 Mouse Mutation Na k atpase Plasmids Rabbit Site directed mutagenesis Vector

Corresponding Organization : Wellcome/MRC Institute of Metabolic Science

Other organizations : University of Liverpool, Karolinska Institutet

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Variable analysis

independent variables

P.Val419Met mutation introduced by site-directed mutagenesis

dependent variables

Expression of KCNG4 with or without a C-terminal HA tag

control variables

KCNG4 cDNA clone
PcDNA3 based expression plasmid (CMV-genex-polioIRESmCherry)
K_V2.1 clone expressing alongside a nuclear GFP reporter in the pCAGGS-IRES2-nucEGFP vector

positive controls

K_V2.1 clone expressing alongside a nuclear GFP reporter in the pCAGGS-IRES2-nucEGFP vector

negative controls

Not specified

Annotations

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