Journal: bioRxiv

Article Title: Molecular Basis of Kv1 Channel Downregulation and Its Epileptogenic Implications in Lgi1 Knock-out Mice

doi: 10.1101/2023.07.24.550266

Figure Lengend Snippet: a) Quantification by Western blot of Kv 2.1 expression in brain homogenates of WT (WT 1.00 ± 0.09) and Lgi1 -/- (1.01 ± 0.11); p = 0.97; n = 15; Two-sample t-test). b) Quantification by Western blot of Kv 2.1 expression in WT (1.00 ± 0.04) and Lgi1 -/- (0.45 ± 0.04) hippocampal extracts; p = 1.00 x 10 -9 ; n = 16; Two-sample t-test). c) 10x representative images showing a reduced expression of Kv 2.1 across the hippocampus of Lgi1 -/- mouse. Scale bar = 100 µm. d) Representative 63x images showing a reduction of Kv 2.1 in CA3 Str. Pyramidale of Lgi1 -/- hippocampus. Scale bar = 5 µm. e) Analysis of Kv 2.1 puncta in CA3 in WT (black) and Lgi1 -/- (red) hippocampi showing a reduction of Kv 2.1 clusters’ density in Lgi1 -/- . (Density of Clusters (#/µm 3 ) WT = 0.036 ± 0.002 Lgi1 -/- = 0.020 ± 0.002; p = 1.53×10 -6 ; Mann-Whitney U test; Surface of clusters (µm 2 ) WT = 3.48 ± 0.09 Lgi1 -/- = 3.27 ± 0.08; p = 0.08; Two sample t-test; Normalized (to WT) immunoreactivity of clusters WT = 1.00 ± 0.01 Lgi1 -/- = 0.99 ± 0.01; p = 0.53; Two sample t-test; Volume of clusters (µm 3 ) WT = 0.16 ± 0.01 Lgi1 -/- = 0.15 ± 0.01; p = 0.07; Two sample t-test). f) Representative 63x images showing a reduction of Kv 2.1 at the level of CA3 Pyr. neurons AIS (CA3 Str. Oriens) of Lgi1 -/- hippocampus. Scale bar = 5 µm. g) 63 Detail of a CA3 Pyr. neuron AIS in WT (top) and Lgi1 -/- (bottom) showing a reduced density of Kv 2.1 clusters at the AIS of Lgi1 -/- neurons. Scale bar = 2 µm. h) Analysis of Kv 2.1 puncta in the AIS of CA3 pyramidal neurons in WT (black) and Lgi1 -/- (red) showing a reduction of Kv 2.1 clusters’ density and volume at the AIS of CA3 pyramidal neurons in Lgi1 -/- mouse. (Density of Clusters (#/µm AIS) WT = 0.96 ± 0.03 Lgi1 -/- = 0.69 ± 0.03; p = 4.58 x 10 -7 ; Two sample t-test; Surface of clusters (µm 2 ) WT = 6.87 ± 0.41 Lgi1 -/- = 5.85 ± 0.27; p = 0.07; Mann-Whitney U test; Normalized (to WT) immunoreactivity of clusters WT= 1.00 ± 0.02 Lgi1 -/- = 0.96 ± 0.01; p = 0.09; Mann-Whitney U test; Volume of clusters (µm 3 ) WT = 0.23 ± 0.01 Lgi1 -/- = 0.18 ± 0.01; p = 0.046; Mann-Whitney U test).

Article Snippet: Guinea pig (386005, RRID: AB_2737033) anti-ankyrinG (AnkG) was from Synaptic Systems and rabbit anti-Kv 2.1 (APC-012) from Alomone labs.

Techniques: Western Blot, Expressing, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inward Rectifier K + Currents Contribute to the Proarrhythmic Electrical Phenotype of Atria Overexpressing Cyclic Adenosine Monophosphate Response Element Modulator Isoform CREM‐IbΔC‐X

doi: 10.1161/JAHA.119.016144

Figure Lengend Snippet: Experimental animals involved wild‐type mice (WT) and transgenic mice (TG) expressing the repressor isoform of cyclic adenosine monophosphate response element modulator, CREM‐IbΔC‐X, and were distributed according to age in 6‐week‐old (WT 6w and TG 6w ) and 12‐week‐old (WT 12w and TG 12w ) groups. A , Representative transient outward K + current (I to ) traces recorded as −40 mV sensitive currents from atrial cardiomyocytes of WT 12w and TG 12w . For clarity, current density (expressed in picoamperes/picofarads [pA/pF]) traces from −40 mV with +20 mV increments are displayed. B , Mean±SE of current–voltage plots of the peak current density (I to ). For WT 6w , n/N are 41/15, for TG 6w n/N are 22/14, for WT 12w n/N are 35/19, and for TG 12w n/N are 20/16. Ca through d , Representative immunoblots and quantification of protein expression of calsequestrin (CSQ; Ca ) normalized to Ponceau and of K + voltage‐gated channel subfamily D member 2 (Kv4.2, Cb ) and 3 (Kv4.3, Cc ), and K + channel interacting protein 2 (KChIP2) ( Cd ) normalized to CSQ. Data show mean±SE of N=7 mice per group, except for Kv4.3, where N=6 as the result of an undetectable band in TG 12w . * P <0.05 TG 6w vs WT 6w (gray), TG 12w vs WT 12w (black), # P <0.05 WT 12w vs WT 6w (gray), TG 12w vs TG 6w (black) from 2‐way repeated‐measures analysis of variance with Bonferroni post hoc test ( B ) and parametric or nonparametric 1‐way analysis of variance with the corresponding post hoc test ( C ).

Article Snippet: The following rabbit polyclonal primary antibodies were used against K + voltage‐gated channels (Kv) subunits Kv4.2 (1:200, APC 023, Alomone Labs, Jerusalem, Israel), Kv4.3 (1:200, APC 017, Alomone Labs), K + channel interacting protein 2 (KChIP2) (1:200, sc‐25685, Santa Cruz Biotechnology Inc, Santa Cruz, CA), and K + inwardly rectifying channel (Kir) subunits Kir2.1 (1:200, APC 159, Alomone Labs), Kir2.3 (1:1000, APC 032, Alomone Labs), Kir3.1 (1:200, APC 005, Alomone Labs), Kir3.4 (1:200, APC 027, Alomone Labs), and as loading control calsequestrin (1:2500, PA1‐913, Thermo Fisher Scientific).

Techniques: Transgenic Assay, Expressing, Western Blot