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: International Journal of Molecular Sciences

Article Title: Deletion of Notch3 Impairs Contractility of Renal Resistance Vessels Due to Deficient Ca 2+ Entry

doi: 10.3390/ijms232416068

Figure Lengend Snippet: Comparative real-time PCR mRNA expression of 47 genes related to contractile function in the afferent arterioles from Notch3 −/− and wild-type littermates. Note that the expression of Cacna1h coding the α 1H subunit of the T-type Ca 2+ channel (Ca v 3.2) gene was strongly downregulated in Notch3 −/− ( p < 0.001 vs. wild-type). In contrast, Cacna1c coding the α 1C subunit of the L-type Ca 2+ channel was similarly expressed in the two strains (No. 26 on the list).

Article Snippet: 11 , IP3-receptor , Ip3r1 , Itpr1-Mm00439917_m1 , 0.68 ± 0.02 , 0.62 ± 0.02 , −0.12 , 0.05.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Journal of Neuroinflammation

Article Title: ‘Medusa head ataxia’: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/ITPR1 and anti-CARP VIII

doi: 10.1186/s12974-015-0356-y

Figure Lengend Snippet: Selected antibodies to cerebellar antigens reported in patients with cerebellar ataxia

Article Snippet: Fig. 6 Expression of ITPR1 in the human cerebellum as demonstrated by IHC using an affinity-isolated rabbit antibody to human ITPR1 (Atlas antibodies, HPA016487).

Techniques: Virus

Journal: Journal of Neuroinflammation

Article Title: ‘Medusa head ataxia’: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/ITPR1 and anti-CARP VIII

doi: 10.1186/s12974-015-0356-y

Figure Lengend Snippet: Expression of ITPR1 in the human cerebellum as demonstrated by IHC using an affinity-isolated rabbit antibody to human ITPR1 (Atlas antibodies, HPA016487). Modified image from the Human Protein Atlas image database

Article Snippet: Fig. 6 Expression of ITPR1 in the human cerebellum as demonstrated by IHC using an affinity-isolated rabbit antibody to human ITPR1 (Atlas antibodies, HPA016487).

Techniques: Expressing, Isolation, Modification

Journal: Journal of Neuroinflammation

Article Title: ‘Medusa head ataxia’: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 1: Anti-mGluR1, anti-Homer-3, anti-Sj/ITPR1 and anti-CARP VIII

doi: 10.1186/s12974-015-0356-y

Figure Lengend Snippet: Binding of IgG from a patient with ITPR1-Ab-positive ACA as determined in a recombinant cell-based assay to formalin-fixed rat cerebellum tissue. Human IgG was detected using a goat anti-human IgG secondary antibody labelled with Alexa Fluor®488 (green fluorescence)

Article Snippet: Fig. 6 Expression of ITPR1 in the human cerebellum as demonstrated by IHC using an affinity-isolated rabbit antibody to human ITPR1 (Atlas antibodies, HPA016487).

Techniques: Binding Assay, Recombinant, Cell Based Assay, Fluorescence

Journal: eLife

Article Title: 5-Hydroxymethylcytosine-mediated active demethylation is required for mammalian neuronal differentiation and function

doi: 10.7554/eLife.66973

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Itpr1 (mouse monoclonal) , Origene , Origene: TA326547 , (1:500).

Techniques: Generated, Recombinant, Construct, Sequencing, Methylation Sequencing

Journal: eLife

Article Title: 5-Hydroxymethylcytosine-mediated active demethylation is required for mammalian neuronal differentiation and function

doi: 10.7554/eLife.66973

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Itpr1 (mouse monoclonal) , Origene , Origene: TA326547 , (1:500).

Techniques:

Journal: bioRxiv

Article Title: A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization

doi: 10.1101/2024.01.03.574069

Figure Lengend Snippet: A . Quantitative PCR showed chromaffin cells express all 3 of the IP3 receptor isoforms, but only 2 RyR isoforms. Mean transcript levels of IP3R1 (0.05 ± 0.003) were approximately 4-fold higher than IP3R2 (0.01 ± 0.002) and almost 10-fold higher than IP3R3 (0.005 ± 0.004). Expression levels were compared to β-actin. B . IP3R and RyR isoforms showed a similar pattern of expression in WT and PLCε KO cells. C and D . Western blot analysis comparing the protein expression levels of IP3R1 in WT and PLCε KO cells. The intensity of IP3R1 bands (WT vs KO) were quantified relative to β-actin and were not significantly different (Student’s t-test; p = 0.6).

Article Snippet: The scrambled shRNA and IP3R1 shRNA plasmids were purchased from OriGene; the 29mer sequence of scrambled shRNA (Cat# TR30015) is 5’-GCACTACCAGAGCTAACTCAGATAGTACT-3’, the 29mer sequence of IP3R1 (also called Itpr1) shRNA (Cat# TF517036) is 5’-TCAGCACCTTAGGCTTGGTTG-ATGACCGT-3’, and the transfected cells were identified based on their red fluorescence encoded by shRNA vectors.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: bioRxiv

Article Title: A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization

doi: 10.1101/2024.01.03.574069

Figure Lengend Snippet: A . Confocal images showing immunostaining for the ER marker KDEL (green) and IP3R1 (red) in a chromaffin cell. Nuclei labeled with DAPI (blue). B and C . KDEL and IP3R1 intensities along the lines were similar. D . Manders’ coefficient analysis for the colocalization of KDEL and IP3R1 in n = 25 cells. Coefficient for IP3R1 overlap with KDEL = 0.7 ± 0.02; coefficient for KDEL overlap with IP3R1 = 0.8 ± 0.02.

Article Snippet: The scrambled shRNA and IP3R1 shRNA plasmids were purchased from OriGene; the 29mer sequence of scrambled shRNA (Cat# TR30015) is 5’-GCACTACCAGAGCTAACTCAGATAGTACT-3’, the 29mer sequence of IP3R1 (also called Itpr1) shRNA (Cat# TF517036) is 5’-TCAGCACCTTAGGCTTGGTTG-ATGACCGT-3’, and the transfected cells were identified based on their red fluorescence encoded by shRNA vectors.

Techniques: Immunostaining, Marker, Labeling

Journal: bioRxiv

Article Title: A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization

doi: 10.1101/2024.01.03.574069

Figure Lengend Snippet: A . Western blot showing IP3R1 immunoreactive bands in control (scrambled shRNA) and IP3R1 KD samples. B . Intensity IP3R1 was normalized to β-actin expression. Relative intensities of bands (means ± SEM) of scrambled = 1 ± 0.007 and KD = 0.4 ± 0.06. Differences were statistically significant (Student’s t-test; p = 0.01). C . Representative traces of PACAP-stimulated Cal-520 fluorescence changes in control (untransfected) cells, scrambled shRNA, IP3R1 KD cells. D . Scatter plot of total spike area (n=49 control; n=17 scrambled shRNA; n=29 IP3R1 shRNA) represented as medians ± interquartile range. A Kruskal-Wallis test was used to assess statistical significance of differences. Control vs IP3R1 KD, p = 2×10 -9 ; IP3R1 KD vs scrambled, p = 0.0003. E . Scatter plot of max amplitude are represented as medians ± interquartile range. A Kruskal-Wallis test was used to assess statistical significance of differences. Control vs IP3R1 KD, p = 7×10 -9 ; IP3R1 KD vs scrambled, p = 3×10 -5 . F . FFN511 release was measured in cells stimulated by PACAP. Responses were reported as fusion events per unit area. n=12 cells for scrambled shRNA and n=13 cells for IP3R1 shRNA. Differences between groups were statistically significant (Mann-Whitney, p = 7×10 -7 ).

Article Snippet: The scrambled shRNA and IP3R1 shRNA plasmids were purchased from OriGene; the 29mer sequence of scrambled shRNA (Cat# TR30015) is 5’-GCACTACCAGAGCTAACTCAGATAGTACT-3’, the 29mer sequence of IP3R1 (also called Itpr1) shRNA (Cat# TF517036) is 5’-TCAGCACCTTAGGCTTGGTTG-ATGACCGT-3’, and the transfected cells were identified based on their red fluorescence encoded by shRNA vectors.

Techniques: Western Blot, Control, shRNA, Expressing, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: A PACAP-activated network for secretion requires coordination of Ca 2+ influx and Ca 2+ mobilization

doi: 10.1101/2024.01.03.574069

Figure Lengend Snippet: A and B. Scatter plot of total spike area and maximum %ΔF/F of Cal520 fluorescence. Cells were stimulated by 1 μM DMPP. n=51 control (untransfected), n=12 for scrambled shRNA, n=27 for IP3R1 KD. Data are shown as medians ± interquartile range. Differences between groups were not statistically significant (Kruskal-Wallis; p > 0.05).

Article Snippet: The scrambled shRNA and IP3R1 shRNA plasmids were purchased from OriGene; the 29mer sequence of scrambled shRNA (Cat# TR30015) is 5’-GCACTACCAGAGCTAACTCAGATAGTACT-3’, the 29mer sequence of IP3R1 (also called Itpr1) shRNA (Cat# TF517036) is 5’-TCAGCACCTTAGGCTTGGTTG-ATGACCGT-3’, and the transfected cells were identified based on their red fluorescence encoded by shRNA vectors.

Techniques: Fluorescence, Control, shRNA

Journal: Frontiers in Molecular Neuroscience

Article Title: Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease

doi: 10.3389/fnmol.2013.00042

Figure Lengend Snippet: List of genes and their annotations included in the RT-qPCR array .

Article Snippet: Itpr1 , inositol 1,4,5-trisphosphate receptor 1 , Mm00439907_m1.

Techniques: Binding Assay