Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Domain structures of short and long Ank2 protein variants (220 and 440 kDa, respectively). Note that exon 4 deletion (red star) affects both short and long variants. MBD membrane-binding domain, SBD spectrin-binding domain; linker, a linker for the MBD and SBD domains; giant exon, a domain uniquely present in the long Ank2 variant; DD death domain, CT C-terminal region. b Immunoblot analysis of WT and Ank2-cKO cortical and hippocampal lysates (P21). ( n = 4 mice [WT, cKO], one sample t -test). c Hyperactivity of Ank2-cKO mice (P19–21) in the open-field test (100 lux), as shown by distance moved. The reduced time spent in the center region suggests anxiety-like behavior. ( n = 25 [WT; males and females mixed here and in all other behavioral experiments], 19 [cKO], Mann-Whitney test [total distance moved, center time], two-way repeated-measures/RM-ANOVA with Sidak’s test [distance moved]). d Anxiolytic-like behavior in Ank2-cKO mice (P19–21) in the light-dark test (600 lux), as shown by light-chamber time/entry. ( n = 20 [WT], 17 [cKO], Student’s t -test). e Anxiolytic-like behavior in Ank2-cKO mice (P24–25) in the elevated plus-maze test, as shown by open-arm time/entry. ( n = 18 [WT], 10 [cKO], Student’s t -test). f Normal social interaction and impaired social novelty recognition in Ank2-cKO mice (P19–20) in the three-chamber test, as shown by sniffing time. S1/S2, novel/familiar stranger; O, object. ( n = 18 [WT], 14 [cKO], Mann–Whitney test [WT-S1/O, WT-S1/S2, and cKO-S1/O], Student’s t-test [cKO-S1/S2]). g Abnormally increased social interaction in Ank2-cKO mice (P23–25) in a direct social-interaction test. ( n = 9 [WT], 7 [cKO], Student’s t -test). h Decreased self-grooming and digging, without a change in jumping, in Ank2-cKO mice (P25–27). Note that hyperactivity is observed here, similar to the open-field hyperactivity. ( n = 24 [WT], 17 [cKO], Mann-Whitney test [digging/self-grooming]). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, Binding Assay, Variant Assay, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of epileptiform spike discharges in Ank2-cKO mice (P35; parietal right lobe) monitored until death, as shown by electroencephalography (EEG). b Epileptiform discharges in EEG observed in an Ank2-cKO mouse until death. c Juvenile seizure-related death occurring in Ank2-cKO mice during ~P20–50. ( n = 23 [WT], 23 [cKO], Log-rank test). d – h Increased pentylenetetrazole (PTZ)-induced seizures in Ank2-cKO mice (P25–27), as shown by final seizure stage (1–4) reached, latency to stage 2 seizure, animals reached stage 2, and frequency and total duration of stage 2. The stage 2 was mainly compared because of its predominance in the mutant mice. ( n = 17 [WT], 10 [cKO], chi-square test [final seizure stage], Mann-Whitney test [stage 2 latency/duration]). The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Examples of neuronal firing in the somatosensory cortex (SSC) in brain slices from Ank2-cKO mice, as measured by multielectrode array (MEA; 64 electrodes in 8 × 8 arrays; cutoff for significant firing > 4.5 x standard deviation). b , c Increased mean firing rate and mean burst rate (inter-spike-interval <10 ms) in the Ank2-cKO SSC (P19–22), as measured by MEA. ( n = 9 slices/3 mice [WT], 10, 3 [cKO], Student’s t -test [total], Welch’s test [layer 2/3], Mann–Whitney test [layer 5]). d Unaltered number of spikes in a single burst in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t-test [total], Mann–Whitney test [layer 2/3 and 5]). e Unaltered percentage of spikes in bursts in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Welch’s test [total], Mann-Whitney test [layer 2/3 and 5]). f Diagram showing examples of local field potentials (LFPs) and depicting parameters of LFP (length and amplitude). g – i Increased duration but normal frequency and amplitude of LFPs in layer 2/3 but not layer 5 in the Ank2-cKO SSC (P19–22) measured by MEA. ( n = 9, 3 [WT], 10, 3 [cKO], Student’s t -test [LFP frequency-layer 2/3, amplitude-total and layer 2/3, duration-total and layer 5], Mann–Whitney test [frequency/amplitude-layer 5], Welch’s test [duration-layer 2/3]). The statistical tests involved two-sided analyses. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Standard Deviation, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Normal resting membrane potential in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–22). ( n = 12 neurons/8 mice [WT], 22,8 [cKO], Mann–Whitney test). b , c Increased input resistance in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 10, 3 [WT], 12, 3 [cKO], two-way RM-ANOVA with Sidak’s test and Student’s t -test). d Increased current-firing curve slope in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 7, 3 [WT], 9, 3 [cKO], two-way RM-ANOVA with Sidak’s test). e Diagram explaining mAHP. f Decreased mAHP amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–22). ( n = 16, 5 [WT], 21, 5 [cKO], Welch’s test). g Diagram explaining AP threshold and AP shape-related parameters. h – l Normal AP threshold and AP shape-related parameters in Ank2-cKO SSC layer 2/3 neurons (P19–22), as indicated by AP-threshold voltage and the height, width, and time-dependent voltage changes (dV/dt; maximal/depolarizing and minimal/repolarizing) of APs. ( n = 13, 9 [WT], 18, 11 [cKO], Student’s t -test [AP threshold and Max dV/dt and min dV/dt], Mann-Whitney test [FWHM and amplitude]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Membrane, MANN-WHITNEY

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a Diagram depicting procedures of PTM (posttranslational modification) analyses for samples from WT and Ank2-cKO cortex and hippocampus; (P19–22) ( n = 3 groups from 9 mice [3 mice/group] [WT, cKO]). b , c DAVID gene ontology (GO) analyses of biological functions for the proteins with up- and downregulated PTM levels (PTM proteins). d – f Volcano plots highlighting the PTM proteins with significant fold changes (>1.5) and p -values (<0.05, Welch’s t-test) and the presence of potassium channels in the down-PTM proteins (fold change > 1.5 and p < 0.05 [Welch’s t -test]). g Diagram depicting procedures of proteomic analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex + hippocampus; P19–22). h , i Volcano plots showing the differentially expressed proteins (DEPs) derived from the analysis of total (whole-lysate) proteins in Ank2-cKO mice (cortex and hippocampus; P19–22) with significant fold changes (>1.5) and p -values (<0.05) and the presence of Kv7.2 and Kv7.3 in the downregulated total DEPs ( p < 0.05 [Welch’s t -test] but not fold change > 1.5). ( n = 3 mice [WT, cKO]). j Decreased total and crude synaptosomal levels of Kv7.2 and Kv7.3 in Ank2-cKO brains (cortex + hippocampus; P19–23). Note that the levels of Kv2.1, Kv3.1, and Kv1.2 (unrelated control) were not changed. ( n = 4 mice [WT, cKO] except for Kv7.3 in total lysates ( n = 5 mice [WT, cKO]), one sample t -test). k Decreased surface levels of Kv7.2 and Kv7.3 in cultured cortical neurons (DIV 17). ( n = 15 dishes [5 + 5 + 5] from 3 biologically independent experiments [Kv7.2] and 14 dishes [5 + 5 + 4] from 3 biologically independent experiments [Kv7.3], one sample t-test). Source data for uncropped immunoblot images are provided as a Source Data file. The statistical tests involved two-sided analyses. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Modification, Derivative Assay, Control, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a – c Increased AIS area (marked by Ank3; arrows) in cultured hippocampal Ank2 fl/fl neurons infected with AAV-Cre (DIV 4 ~ 10–14 ~ 28), and decreased mean intensities of Kv7.3 and Ank3 in the AIS area; these changes together yield normal total intensities of Kv7.3 and Ank3. Note that the width of Ank3 was not changed. ( n = 36 neurons from three independent experiments [WT], 36 [cKO], Welch’s test [Ank3 mean intensity], Mann–Whitney test [Ank3 area/total intensity; Kv7.3 mean/total intensity in AIS]). Scale bar, 10 µm. d – g Increased AIS length and area (marked by Ank3) in the Ank2-cKO somatosensory cortex (P19–23), as determined by three-dimensional imaging and quantification of expanded brain tissues. ( n = 60 neurons from 4 mice [WT], 60, 4 [cKO], Student’s t-test [Ank3 length], Welch’s t -test [Ank3 area]). Scale bar, 4 µm. h – j The Kv7.2/3 antagonist, XE991, increases the slope of the current-spike curve in WT, but not Ank2-cKO, SSC layer 2/3 pyramidal neurons (P19–23). ( n = 7 neurons from 3 mice [WT], 14,4 [WT + XE991], 9,3 [cKO], 13,5 [cKO+XE991], two-way RM-ANOVA with Sidak’s test [i] and with Tukey’s test [j]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Cell Culture, Infection, MANN-WHITNEY, Imaging

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Retigabine treatment (from P16–17) rescues the increased neuronal excitability and firing in Ank2-cKO SSC layer 2/3 pyramidal neurons (P19–23) without affecting WT neurons, as shown by current-firing curves. ( n = 8 neurons from 4 mice [WT-Veh], 8,4 [WT-RTG], 8,4 [cKO-Veh], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [a], two-way RM-ANOVA with Sidak’s test [b]). c and d Retigabine treatment (from P16–17) rescues the decreased mAHP (medium afterhyperpolarization) amplitude in Ank2-cKO SSC layer 2/3 neurons (P19–23) without affecting that of WT neurons. ( n = 8, 4 [WT], 8,4 [WT-RTG], 8,4 [cKO], 8,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [c], two-way RM-ANOVA with Sidak’s test [d]). e , f Retigabine treatment (from P16–17) does not affect input resistance in Ank2-cKO or WT SSC layer 2/3 neurons (P19–23). ( n = 7,4 [WT], 7,4 [WT-RTG], 7,4 [cKO], 7,4 [cKO-RTG], two-way RM-ANOVA with Tukey’s test [e], two-way RM ANOVA with Sidak’s test [f]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/- SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques:

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet: a , b Chronic retigabine treatment (from P16–17) improves juvenile seizure-related death of Ank2-cKO mice without affecting WT mice. ( n = 11 mice [WT-Veh/vehicle], 12 [WT-RTG/retigabine], 16 [cKO-Veh], 10 [cKO-RTG], Log-rank test). c , d Cessation of chronic retigabine treatment at around ~P42 (~26 days after the initiation of retigabine treatment) eliminates the treatment effect of retigabine on juvenile seizure-related death in Ank2-cKO mice. ( n = 14 mice [cKO-Veh], 13 [cKO-RTG], Log-rank test). e – g Chronic retigabine treatment (from P16–17) does not improve hyperactivity or anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. ( n = 11 mice [WT-Veh], 12 [WT-RTG], 11 [cKO-Veh], 10 [cKO-RTG], two-way ANOVA with Tukey test). h – k Acute retigabine treatment (5 mg/kg) at P21/22 improves hyperactivity but not anxiety-like behavior (center time) of Ank2-cKO mice in the open-field test. Drug treatment groups were divided into two (retigabine/saline first) to minimize cross-treatment effects. ( n = 13 mice [WT-Veh], 15 [WT-RTG], 16 [cKO-Veh], 17 [cKO-RTG], two-way ANOVA with Tukey’s test [distance moved_4min], Sidak’s test [distance moved_total]). The statistical tests involved two-sided analyses, and adjustments were made for multiple comparisons. Data are presented as mean values +/− SEM. P -values in figure panels: * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Saline

Journal: Nature Communications

Article Title: Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice

doi: 10.1038/s41467-023-39203-z

Figure Lengend Snippet:

Article Snippet: The supernatants were incubated with 1 µg of primary antibodies (mouse IgG, anti-Ank2 antibody, StressMarq, AB_11232606; anti-Ank3 antibody, Santa Cruz Biotechnology, sc-12719) at 4 °C for 2 h under rotator.

Techniques: Virus, Recombinant, Software

Journal: bioRxiv

Article Title: Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes

doi: 10.1101/2020.04.30.071076

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CamKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for Kcc2 and components of the spectrin/ankyrin subnetwork; Sptan1, Sptbn1, Ank2, Itpr1. Colocalization was measured using line scans (white lines), and represent the normalised fluorescent intensity of single pixels (approximately 1 μm) on dendrites (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (Icc): Ank2 (mouse, Icc, Invitrogen 33-3700), Ank2 (rabbit, IB, Bioss BS-6967R-TR), Cntn1 (rabbit, IB), Abcam Ab66265), Itpr1 (rabbit, IB/Icc, Alomone Acc-019), Kcc2 (mouse, IP/Icc, Neuromab 75-013), Kcc2 (rabbit, IB/Icc, Millipore 07-432), Scn2a (mouse, Neuromab 75-024), Scn2a (rabbit, IB, Alomone ASC-002), Sptan1 (mouse, Icc, Abcam Ab11755), Sptan1 (rabbit, IB, Cell Signaling 21225), Sptbn1 (rabbit, IB/Icc, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 protein complexes were isolated from forebrain plasma membrane fractions of 8-12-week-old mice, resolved by BN-PAGE and immunoblotted for selected high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CAMKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for KCC2 and high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1 (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Total forebrain lysates and plasma membrane lysates were resolved by SDS-PAGE and immunoblotted for selected high risk ASD/Epi risk gene product; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1. B. The expression levels for each protein were quantified using densitometry and normalized to α-Tubulin loading controls (n=3). KCC2 (p=0.037), ANK3-190 (p=0.0014), CNTN1 (p=0.011), and ITPR1 (p=0.016) were significantly reduced in plasma membrane fractions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Clinical Proteomics, Membrane, SDS Page, Expressing

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Differential centrifugation of homogenized forebrain from 8-12-week-old mice was used to fractionate cellular organelle and obtain an enriched plasma membrane fraction. B. Western blots of the organelle fractions were used to measure the abundance of organelle markers; HSP90 (cytosol), HSP60 (mitochondria), calreticulin (ER/Golgi), n-Cadherin (n-Cadh) (plasma membrane). KCC2 abundance was also measured to confirm KCC2 enrichment in the plasma membrane fraction. C. Blue Native PAGE (BN-PAGE) was carried out on plasma membrane lysates, and immunoprecipitated KCC2 to resolve the native protein complexes that contain KCC2. These were correlated with protein bands observed by Coomassie staining. D. Detected peptides were mapped to the KCC2 reference sequence to determine KCC2 sequence coverage obtained from LC-MS/MS of BN-PAGE protein bands produced following KCC2 IP. Bar charts of the KCC2 sequence coverage expressed as a percentage of the full sequence and by total KCC2 peptides detected (n=4).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Centrifugation, Clinical Proteomics, Membrane, Western Blot, Blue Native PAGE, Immunoprecipitation, Staining, Sequencing, Liquid Chromatography with Mass Spectroscopy, Produced

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Pie charts showing the average number of total KCC2 peptides detected by LC-MS/MS relative to the average number of total peptides detected for all other proteins in each molecular weight complex. B. Venn diagrams showing the number and overlap of KCC2-associated proteins identified in each of 4 biological replicates in each molecular weight complex. C. Venn diagram showing the overlap in associated proteins detected in at least 3 of the 4 replicates for each molecular weight complex. D. PCA analysis of each biological replicate for each molecular weight complex based on their protein composition.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Liquid Chromatography with Mass Spectroscopy, Molecular Weight

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. A network diagram including the 150 most abundant proteins from the 600 kDa complex based on total peptide counts. Known interactions were obtained using stringent high confidence, direct experimental association parameters from StringDB. These were used to construct a network diagram of protein nodes and arrows to indicate known interactions. The interactions for each protein with KCC2 were included as discovered here. An overlay of gene ontology (GO) terms was used to provide protein classification information. B. A network diagram for the most abundant 150 proteins in the 800 kDa complex, analyzed as described above (n=4).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Construct

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. A network diagram of KCC2-associated proteins from the 600 kDa complex, that are also the protein products of genes associated with ASD or epilepsy. The known protein associations were sourced from StringDB and applied with an overlay of ASD/Epi risk genes from the SFARI, and EpilepsyGene databases. The colors indicate whether the proteins are implicated in ASD or epilepsy alone, or both. B. The same analysis was carried out for the 800 kDa protein complex (n=4).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques:

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 protein complexes were isolated from forebrain plasma membrane fractions of 8-12-week-old mice, resolved by BN-PAGE and immunoblotted for selected high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CAMKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for KCC2 and high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1 (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Total forebrain lysates and plasma membrane lysates were resolved by SDS-PAGE and immunoblotted for selected high risk ASD/Epi risk gene product; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1. B. The expression levels for each protein were quantified using densitometry and normalized to α-Tubulin loading controls (n=3). KCC2 (p=0.037), ANK3-190 (p=0.0014), CNTN1 (p=0.011), and ITPR1 (p=0.016) were significantly reduced in plasma membrane fractions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Clinical Proteomics, Membrane, SDS Page, Expressing

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 was isolated from plasma membrane fractions of mouse forebrain and resolved by SDS-PAGE. B. Bar graphs of high confidence phosphorylated peptides relative to total detected peptides in wild type and FMR1 KO mice. For bars where phosphorylation was detected, A scores are superimposed to express phosphoryation confidence levels. C. Diagram of high confidence KCC2 phosphorylation site positions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation, Clinical Proteomics, Membrane, SDS Page, Phospho-proteomics

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 protein complexes were isolated from forebrain plasma membrane fractions of 8-12-week-old mice, resolved by BN-PAGE and immunoblotted for selected high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CAMKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for KCC2 and high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1 (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Total forebrain lysates and plasma membrane lysates were resolved by SDS-PAGE and immunoblotted for selected high risk ASD/Epi risk gene product; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1. B. The expression levels for each protein were quantified using densitometry and normalized to α-Tubulin loading controls (n=3). KCC2 (p=0.037), ANK3-190 (p=0.0014), CNTN1 (p=0.011), and ITPR1 (p=0.016) were significantly reduced in plasma membrane fractions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: SDS Page, Expressing

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 protein complexes were isolated from forebrain plasma membrane fractions of 8-12-week-old mice, resolved by BN-PAGE and immunoblotted for selected high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation, Clinical Proteomics, Membrane

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CAMKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for KCC2 and high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1 (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Total forebrain lysates and plasma membrane lysates were resolved by SDS-PAGE and immunoblotted for selected high risk ASD/Epi risk gene product; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1. B. The expression levels for each protein were quantified using densitometry and normalized to α-Tubulin loading controls (n=3). KCC2 (p=0.037), ANK3-190 (p=0.0014), CNTN1 (p=0.011), and ITPR1 (p=0.016) were significantly reduced in plasma membrane fractions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Clinical Proteomics, Membrane, SDS Page, Expressing

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. KCC2 protein complexes were isolated from forebrain plasma membrane fractions of 8-12-week-old mice, resolved by BN-PAGE and immunoblotted for selected high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Isolation

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Primary cultured neurons from P1 pups were infected with CAMKII AAV-GFP at DIV 3 (to visualize cell morphology and to identify excitatory neurons) and fixed at DIV 21. The cells were immunostained for KCC2 and high risk ASD/Epi risk gene products; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1 (n=3).

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: Cell Culture, Infection

Journal: bioRxiv

Article Title: The K-Cl co-transporter 2 is a point of convergence for multiple autism spectrum disorder and epilepsy risk gene products

doi: 10.1101/2020.03.02.973859

Figure Lengend Snippet: A. Total forebrain lysates and plasma membrane lysates were resolved by SDS-PAGE and immunoblotted for selected high risk ASD/Epi risk gene product; ANK2, ANK3, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1 and SPTBN1. B. The expression levels for each protein were quantified using densitometry and normalized to α-Tubulin loading controls (n=3). KCC2 (p=0.037), ANK3-190 (p=0.0014), CNTN1 (p=0.011), and ITPR1 (p=0.016) were significantly reduced in plasma membrane fractions.

Article Snippet: The following antibodies were used for immunoprecipitation (IP), immunoblot (IB), or immunocytochemistry (ICC): ANK2 (mouse, ICC, Invitrogen 33-3700), ANK2 (rabbit, IB, Bioss BS-6967R-TR), ANK3 (mouse, ICC, Neuromab 75-146), ANK3 (rabbit, IB, Synaptic Systems 386003), CNTN1 (rabbit, IB/ICC, Abcam Ab66265), ITPR1 (rabbit, IB/ICC, Alomone ACC-019), KCC2 (mouse, IP/ICC, Neuromab 75-013), KCC2 (rabbit, IB/ICC, Millipore 07-432), NCKAP1 (rabbit, IB, Abcam 126061), NCKAP1 (rabbit, ICC, Sigma HPA020449), SCN2A (mouse, ICC, Neuromab 75-024), SCN2A (rabbit, IB, Alomone ASC-002), SHANK3 (rabbit, IB/ICC, Alomone APZ-013), SPTAN1 (mouse, ICC, Abcam Ab11755), SPTAN1 (rabbit, IB, Cell Signaling 21225), SPTBN1 (rabbit, IB/ICC, Abcam Ab72239), α-Tubulin (mouse, IB, Sigma T9026).

Techniques: SDS Page, Expressing