Ab22897

To investigate expression of the KCNQ2 protein in the brain, 4-week-old Y284C heterozygous (n = 3) and homozygous mice (n = 3) and their wildtype male littermates (n = 3) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and transcardially perfused with 0.1 M phosphate-buffer followed by 4% paraformaldehyde in 0.1 M phosphate-buffer. The brains were quickly removed, immersed in the same fixative over night, and stored in 30% sucrose containing 0.1 M phosphate-buffer at 4°C. Vibratome sections (50-µm thick) were cut from the frontal cortex/striatum and hippocampus/thalamus, immersed in 1% H₂O₂ for 15 min, and blocked with 5% normal goat serum for 30 min. The sections were then incubated with polyclonal rabbit anti-KCNQ2 antibody (1∶250, ab22897; Abcam, UK) for 24 h at 4°C, followed by incubation with a biotinylated secondary antibody (1∶200; Vector Laboratories, Burlingame, CA) for 1 h and avidin-biotin peroxidase complex (1∶200; Vector Laboratories) for 1 h. The reaction was developed with diaminobenzidine (0.1 mg/mL) containing 0.0015% H₂O₂. For quantitative analyses, cells exhibiting somatic staining were defined immunopositive. In each mouse, we counted the number of immunopositive cells in layers II/III and V of the frontal cortex and expressed the results as cell number per unit area (1 mm²).

Rat brain membranes (RBMs) and cell lysates from tsA201 cells transiently expressing KCNQ2/pECFP‐N1 were prepared as described previously (Foster et al. 2012). RBMs and cell lysates were solubilized in 1% Triton X‐100 lysis buffer and incubated overnight with a rabbit anti‐KCNQ2 (AB22897, Abcam, Cambridge, UK; 1–2 μg ml⁻¹) or a mouse anti‐KCNQ2 (N26A/23, NeuroMab, Davis, CA, USA; 1–2 μg ml⁻¹), and rabbit anti‐GFP polyclonal antibodies (A6455, Thermo Fisher; 1–2 μl ml⁻¹), respectively. Antigen–antibody complexes were then incubated with protein G beads (GE Healthcare, Uppsala, Sweden) for 4 h at 4°C. After six washes, bound proteins were eluted in SDS sample buffer at 95°C for 3 min. Eluted proteins were size fractionated on SDS‐PAGE gels, and visualized by a colloidal blue staining (Thermo Fisher). For immunoblot with GST‐Kv7.2C, purified proteins were separated on SDS‐PAGE gels and transferred to polyvinylidene fluoride membranes (Waters, Milford, MA, USA), which were then immunostained with anti‐KCNQ2 (N26A/23, 0.22 μg ml⁻¹) antibody.

For SGK1.1 detection we used a rabbit polyclonal antibody produced in our laboratory (Martin-Batista et al., 2021 (link)). SGK1 was detected using rabbit anti-SGK1 polyclonal antibody (Abcam, ab43606). Kv7.2 was detected with rabbit anti-Kv7.2 polyclonal antibody (Abcam, ab22897). YFP-tagged SGK1.1 and GFP-tagged Kv7 subunits were detected using mouse anti-GFP monoclonal antibody (Abcam, ab290). Nedd4-2 was detected with a rabbit polyclonal antibody from Cell Signaling (4013S). GAPDH was detected with a mouse monoclonal antibody (Abcam, 9484). Secondary antibodies conjugated to fluorophores were obtained from Thermo Fisher Scientific (Alexa 594, A-11042; Alexa-488, A-11008).