guinea pig anti-kcnn2 antibody  (Alomone Labs)

Journal: Scientific Reports

Article Title: PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

doi: 10.1038/s41598-020-66790-4

Figure Lengend Snippet: Effects of TBS and FSK treatment on the number of SK2-positive synapses in both WT and AS mice. ( a ) Representative images of SK2 (red) and PSD95 (green) co-immunostaining in the CA1 region of WT and AS mice after baseline recording (used as control) and 40 min after TBS applications with or without FSK treatment. Scale bar, 5 µm. ( b ) Quantitative results (mean ± SEM) of synapses dually labeled by anti-SK2 and anti-PSD95 antibodies in different experimental groups (p = 0.0163, WT Baseline vs WT TBS, p = 0.3209, WT Baseline vs WT FSK, p = 0.0022, WT Baseline vs WT FSK/TBS, p < 0.001, WT baseline vs AS Baseline, p = 0.001, AS Baseline vs AS TBS, p = 0.0045, AS Baseline vs AS FSK, p < 0.001, AS Baseline vs AS FSK/TBS, p = 0.0098, AS TBS vs AS FSK/TBS, n = 3 slices from 3 mice/group, two-way ANOVA followed by Tukey test).

Article Snippet: The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441).

Techniques: Immunostaining, Labeling

Journal: Scientific Reports

Article Title: PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

doi: 10.1038/s41598-020-66790-4

Figure Lengend Snippet: Effects of PKA inhibition on 2xTBS-induced LTP and synaptic SK2 levels in hippocampal slices of AS mice. ( a ) PKA inhibition with KT5720 blocked 2xTBS-induced LTP in AS mice. ( b ) Representative images of SK2 (red) and PSD95 (green) co-immunostaining in the CA1 region of WT and AS mice after baseline recording (used as control) and 2xTBS applications with or without KT5720 treatment. Scale bar, 5 µm. ( c ) Quantitative results (mean ± SEM) of synapses dually labeled by anti-SK2 and anti-PSD95 antibodies in different experimental groups (p = 0.0017, WT Baseline vs WT 2xTBS, p < 0.001, WT Baseline vs AS Baseline, p < 0.001, AS Baseline vs AS 2xTBS, p < 0.001, AS 2xTBS vs AS KT5720/2xTBS, n = 3 slices from 3 mice/group, one-way ANOVA followed by Tukey test).

Article Snippet: The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441).

Techniques: Inhibition, Immunostaining, Labeling

Journal: Scientific Reports

Article Title: PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

doi: 10.1038/s41598-020-66790-4

Figure Lengend Snippet: SK2 and Rab11 co-immunostaining in hippocampal CA1 region of WT and AS mice at different time points after baseline recording (used as control) and TBS applications with or without FSK treatment. ( a ) Representative images of SK2 (red) and Rab11 (green) co-immunostaining. Scale bar, 5 µm. ( b ) Quantitative results (mean ± SEM) of mean fluorescent intensity (MFI) of SK2 in different experimental groups (p = 0.0267, WT Baseline vs WT TBS 60 min, p = 0.0077, WT Baseline vs WT FSK/TBS 15 min, p < 0.001, WT Baseline vs WT FSK/TBS 60 min, p < 0.001, WT baseline vs AS Baseline, p < 0.001, AS Baseline vs AS TBS 15 min, p = 0.0020, AS Baseline vs AS FSK/Baseline, p < 0.001, AS Baseline vs AS FSK/TBS 15 min, p < 0.001, AS Baseline vs AS FSK/TBS 60 min, p < 0.001, AS TBS 60 min vs AS FSK/TBS 60 min, n = 3 slices from 3 mice/group, two-way ANOVA followed by Tukey test). ( c ) Quantitative results (mean ± SEM) of puncta dually labeled by anti-SK2 and anti-Rab11 antibodies in different experimental groups (p = 0.0361, WT Baseline vs WT TBS 15 min, p = 0.0014, WT Baseline vs WT FSK/TBS 15 min, p = 0.0149, WT Baseline vs WT FSK/TBS 60 min, p < 0.001, WT baseline vs AS Baseline, p = 0.0313, AS Baseline vs AS TBS 15 min, p = 0.0032, AS Baseline vs AS FSK/Baseline, p < 0.001, AS Baseline vs AS FSK/TBS 15 min, p = 0.011, AS Baseline vs AS FSK/TBS 60 min, p = 0.0017, AS TBS 60 min vs AS FSK/TBS 60 min, n = 3 slices from three mice/group, two-way ANOVA followed by Tukey test). See also Supplementary Fig. .

Article Snippet: The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441).

Techniques: Immunostaining, Labeling

Journal: Scientific Reports

Article Title: PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

doi: 10.1038/s41598-020-66790-4

Figure Lengend Snippet: Effects of phosphorylation of SK2 on Ube3a-mediated SK2 ubiquitination and SK2 endocytosis. ( a ) Potential phosphorylation (green SSS) and ubiquitination sites (red Ks) in the C-terminal domain of SK2 channel (shaded sequence indicates calmodulin binding domain). ( b ) Distribution of distance between phosphorylation site S568 and C-terminal residue S580 obtained from 100 ns molecular dynamics simulation of SK2 C-terminal helix in explicit solvent. Blue is the distribution curve without phosphorylation, black is that with phosphorylation on serine 568-570. The representative conformation of the helix with the peak distance of 7 Å for unphosphorylated system and 21 Å for tri-phosphorylated system are shown next to each peak. The overlay of the helix backbone conformations from every 2 ns are shown for each system. Serine 568-570 are shown in licorice. The backbones are colored by residue types: positively charged in blue, negatively charged in red, polar in green, and nonpolar in white. ( c ) In vitro ubiquitination of SK2 and its phosphomimetic mutant SK2-3S/D by recombinant Ube3a. Reaction products were analyzed by Western blot with SK2 antibodies. ( d ) Representative images and quantitative analysis (shown as numbers in red; normalized to the Tac-SK2 group set to 100%; N = 3 independent experiments) of Western blots labeled with ubiquitin (Ub) and SK2 antibodies. Samples from COS-1 cells co-transfected with Tac-SK2 plus Ube3a or ∆Ube3a and treated with either DMSO or forskolin (FSK) and Ro 20-1724 were immunoprecipitated using mouse anti-Tac antibodies and probed with indicated antibodies. ( e,f ) Effects of Ube3a overexpression and S-A or S-D mutations on SK2 surface expression and endocytosis. ( e ) Representative images of internalized (red) or surface-expressed (green) Tac-SK2, 3S/A, and 3S/D in COS-1 cells co-transfected with HA (control; top), HA-Ube3a (middle), or HA-∆Ube3a (bottom). Scale bar, 10 µm. ( f ) Quantitative analysis of images in e. Data are expressed as mean ± SEM. p < 0.001 for Tac-SK2/HA vs Tac-SK2/HA-Ube3a, Tac-SK2/HA vs Tac-SK2/HA-∆Ube3a, Tac-SK2-3S/A/HA vs Tac-SK2-3S/A/HA-Ube3a, Tac-SK2-3S/D/HA vs Tac-SK2-3S/D/HA-∆Ube3a, Tac-SK2/HA vs Tac-SK2-3S/A/HA, Tac-SK2/HA vs Tac-SK2-3S/D/HA, Tac-SK2/HA-Ube3a vs Tac-SK2-3S/D/HA-Ube3a; p = 0.0302 Tac-SK2-3S/A/HA vs Tac-SK2-3S/A/HA-∆Ube3a; two-way ANOVA with Tukey post hoc analysis. N = cells is indicated in each column and from at least 3 independent experiments. See also Supplementary Fig. .

Article Snippet: The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441).

Techniques: Sequencing, Binding Assay, In Vitro, Mutagenesis, Recombinant, Western Blot, Labeling, Transfection, Immunoprecipitation, Over Expression, Expressing

Journal: Scientific Reports

Article Title: PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

doi: 10.1038/s41598-020-66790-4

Figure Lengend Snippet: SK2 expression in hippocampus of Ube3a transgenic (Tg) and wild-type (WT) mice. ( a,b ) Western blot analysis of SK2 levels in crude synaptosomal fractions (P2) from hippocampus of WT and Tg mice. Optical densities of the SK2 band are normalized to those of β-actin. Data are expressed as % of values in WT mice and shown as means ± SEM of 5 mice from at least 3 litters; p = 0.0122 for SK2-long, p = 0.001 for SK2-short (unpaired t test). ( a ) representative Western blot images; ( b ) quantitative analysis of blots in a. ( c,d ) Effects of acute forskolin (FSK) treatment on SK2 levels in hippocampus slices of WT and Tg mice. ( c ) representative Western blot images; ( d ) quantitative analysis of blots in c. N = 6 independent experiments, for SK2-long, p < 0.001 WT/control vs WT/FSK, p < 0.001 WT/control vs Tg/control, p = 0.001 Tg/control vs Tg/FSK; for SK2-short, p < 0.001 WT/control vs WT/FSK, p < 0.001 WT/control vs Tg/control, p = 0.0011 Tg/control vs Tg/FSK, two-way ANOVA with Tukey’s post-test. ( e,f ) Effects of acute FSK treatment on ubiquitination and phosphorylation of SK2 in hippocampus slices of WT and Tg mice. ( e) Representative Western blot images. Immunoprecipitation was performed with anti-SK2 antibodies, and Western blots were labeled with anti-SK2, -phosphoserine, and -ubiquitin (Ub) antibodies. Arrows indicate ubiquitinated and/or phosphorylated SK2 proteins. ( f ) Quantitative analysis of blots in e. N = 5 independent experiments. Left panel, p = 0.0025 WT/control vs WT/FSK, p = 0.0073 Tg/control vs Tg/FSK; Right panel, p = 0.004 WT/control vs Tg/control, p = 0.0335 Tg/control vs Tg/FSK, two-way ANOVA with Tukey’s post-test. ( g) Model for SK2 channel regulation by Ube3a-ubiquitination and PKA-phosphorylation. PKA phosphorylation and Ube3a ubiquitination result in SK2 endocytosis in early endosomes (EE). Ubiquitinated SK2 does not recycle back to synaptic membranes through recycling endosomes (RE). PKA and Ube3a jointly regulate synaptic SK2 levels; phosphorylation facilitates ubiquitination.

Article Snippet: The following primary antibodies were used: Tac7G7 (7G7B6, ATCC, HB-8784), SK2 (Alomone, APC-028), SK2 (Alomone, AGP-045), Ube3a (Sigma, E8655), Ube3a (Bethyl Laboratories, A300–351A), PSD95 (Invitrogen, MA1–045), Rab11 (abcam, ab95375), Ubiquitin (Ub, abcam, ab7780), Ub (Santa Cruz, sc-9133), Phosphoserine (Millipore, ab1603), HA (Sigma, H6908), EEA1 (abcam, ab2900), LAMTOR4 (Cell signaling technology, 12284), and β-actin (Sigma, A5441).

Techniques: Expressing, Transgenic Assay, Western Blot, Immunoprecipitation, Labeling