2.4. Immunoblotting

Forebrain P3 membrane protein fractions were isolated from male and female WT, Kcnb1^C/+ and Kcnb1^C/C mice at P58–83. Membrane fractions (50 μg) were separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. Blots were probed with anti-K_V2.1 (K89/34) and anti-mortalin/GRP75 antibodies (Table 2). Alexa Fluor 790 goat anti-mouse antibodies (Table 2) were used to detect signal on an Odyssey imaging system (LI-COR). Relative protein levels were determined by densitometry using Image Studio (LI-COR) and expressed as a ratio of K_V2.1 to GRP75 with WT normalized to 1 and included 7–14 biological replicates per genotype. Normality of transcript and protein expression was assessed by D’Agostino & Pearson test and statistical comparison between groups was made using the nonparametric test Kruskal-Wallis with Dunn’s post-hoc comparisons (GraphPad Prism).

List of Antibodies

WB, western blot; ICC, immuncytochemistry