Journal: International Journal of Molecular Sciences

Article Title: Augmented K Ca 2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice

doi: 10.3390/ijms222413585

Figure Lengend Snippet: Expression of K Ca 2.3 and IP 3 R in mouse myometrium. The upper left panel shows immunofluorescence staining of K Ca 2.3 channels (red) and IP 3 Rs (green) in a longitudinal strip of mouse myometrium; cell nuclei are blue. The boxed region is expanded (bottom left) to show distinct K Ca 2.3 and IP 3 R staining patterns; insets show zoom of the designated regions. The merged image reveals regions of overlapping K Ca 2.3 and IP 3 R densities. The lower right panel highlights overlapping K Ca 2.3 and IP 3 R-positive signals (yellow); cell nuclei are included for reference. Images are representative of tissue preparations from three animals. Scale bar 10 µm.

Article Snippet: Bovine serum albumin (BSA) was added to block nonspecific binding, and tissue was incubated overnight with K Ca 2.3 primary rabbit antibody (1:300; Alomone Labs Jerusalem, Israel) at 4 °C and IP 3 R primary goat antibody (1:250; MyBioSource, San Diego, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Stripping Membranes, Tissue

Journal: International Journal of Molecular Sciences

Article Title: Augmented K Ca 2.3 Channel Feedback Regulation of Oxytocin Stimulated Uterine Strips from Nonpregnant Mice

doi: 10.3390/ijms222413585

Figure Lengend Snippet: Schematic of proposed internal Ca 2+ store feedback regulation of uterine contractions. Action potentials elicit phasic myometrial contractions by opening smooth muscle VGCCs. GPCR binding by oxytocin normally augments contractions via IP 3 -dependent release of internal (ER) Ca 2+ stores. Concurrently, local Ca 2+ release sites positioned near membrane K Ca 2.3 channels solicit negative feedback control of VGCC influx, and limit overall contraction. Introduction of CyPPA increases the Ca 2+ sensitivity of K Ca 2.3 channels, thereby, potentiating K Ca 2.3 feedback regulation. Heightened sensitization of internal store feedback may effectively block productive contractions in the presence of oxytocin.

Article Snippet: Bovine serum albumin (BSA) was added to block nonspecific binding, and tissue was incubated overnight with K Ca 2.3 primary rabbit antibody (1:300; Alomone Labs Jerusalem, Israel) at 4 °C and IP 3 R primary goat antibody (1:250; MyBioSource, San Diego, CA, USA).

Techniques: Binding Assay, Membrane, Control, Sensitivity, Blocking Assay

Journal: bioRxiv

Article Title: SK3-overexpression drives sex-dimorphic working and social memory impairment in mice

doi: 10.1101/2020.07.31.230656

Figure Lengend Snippet: SK3 overexpression leads to sex-dimorphic working and social memory impairment. A and C- short- and long-term memory tasks performed by male mice in the Novel Objects Recognition Test (NOR); B and D- short- and long-term memory tasks performed by female mice in the NOR (n=10 per group). E-J: results derived from the social box test. E, F, H, and I: pre-test/habituation sessions of male and female, respectively. G and J: Memory retention test of male and female, respectively. (N= 10 per group). Bars represent mean+/− standard error mean (SEM). * represents differences to the WT group whereas # represents differences between trials of the same genotype; p< 0.05.

Article Snippet: similar to the protocol described for the determination of pAkt and total Akt (SK3 antibody; 1:500-Alomone Labs-Jerusalem, Israel)

Techniques: Over Expression, Derivative Assay

Journal: bioRxiv

Article Title: SK3-overexpression drives sex-dimorphic working and social memory impairment in mice

doi: 10.1101/2020.07.31.230656

Figure Lengend Snippet: SK3-overexpression induces an increased number of Type 1 progenitors (SOX2+ GFAP+) and filopodia spines, impaired cell migration, and Neuregulin-1 signaling in MALE mice. A-I: results from the double-labeling analysis of cells expressing SOX2+GFAP in the subgranular zone of the dentate gyrus (DG). J, K, and L: display the results related to doublecortin positive cells migrating towards the granular zone of the DG. Arrows indicate cells in the process of migration (K and L). M-results from the qPCR analysis of the DG of the hippocampus (N=3/group). N-X: results and representative images of the analysis of the number and shape of dendritic spines in the granular neurons of the hippocampus. V and Y: qPCR and WB for Neuregulin (NGR1) and its receptor EBbr4. Bars represent mean+/− SEM. * indicates a difference with respect to the WT group. N=5 per group except for the qPCR analysis).

Article Snippet: similar to the protocol described for the determination of pAkt and total Akt (SK3 antibody; 1:500-Alomone Labs-Jerusalem, Israel)

Techniques: Over Expression, Migration, Labeling, Expressing

Journal: bioRxiv

Article Title: SK3-overexpression drives sex-dimorphic working and social memory impairment in mice

doi: 10.1101/2020.07.31.230656

Figure Lengend Snippet: SK3 overexpression negatively modulates Akt/mTOR and MAPKinase, signaling the DG of male, but not female mice. A and B: pAkt/total Akt expression measures by Western blot (WB) in the DG of female and male mice, respectively. C and D: pmTOR/mTOR; E and F: GβL/α-tubulin; G-H: RAPTOR/α-tubulin; I-J: pERK1/2/GAPDH; pJNK/GAPDH. Representative images of the entire row of the WB membrane and its respective loading control. Bars represent mean+/− SEM. N=4 per group., except for the ERK and JNK in the WT group (n=3). * represents a difference from the WT group; p<0.001.

Article Snippet: similar to the protocol described for the determination of pAkt and total Akt (SK3 antibody; 1:500-Alomone Labs-Jerusalem, Israel)

Techniques: Over Expression, Expressing, Western Blot

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Primary antibodies.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques:

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Characterization of the molecular interactions between Cav1.3, SK3, and HCN channels using co-immunoprecipitation and immunoblot. Proteins were membrane-extracted in CL47a solubilization buffer (IP1) or in CL48 buffer (IP2) and immunoprecipitated using anti-Cav1.3 ( A ), anti-SK3 ( B ), and anti-Kv4.3 antibodies ( C ). Blots were performed on starting material, P2 membrane fraction (P2), or on solubilized P2 proteins (sol) or after immunoprecipitation (IP1, IP2). Blots were probed with anti-SK3 (( A ), right panel), anti-Cav1.3 (( B ), right panel), anti-HCN4 (( A , B ) left panels; ( C ), right panel), anti-Kv4.3 (( C ), left panel), and anti-HCN2 (( C ), middle panel). P2: enriched P2 membrane fraction. IP1: immunoprecipitated proteins in CL47a buffer. IP2: immunoprecipitated proteins in CL48 buffer. sol: solubilisate. WB: Western blot (immunoblot).

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Membrane, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Schematic representation of midbrain ion channel interactions detected through co-immunoprecipitation and immunoblot. Lines indicate co-immunoprecipitated ion channels. The arrows point from the bait to the prey revealed through immunoblot.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Protein nano-environment of midbrain ion channels determined using affinity-purification mass spectrometry. ( A ) Left panel, Cav1.2 subunit ( Cacna1c gene) immunoprecipitations. Right panel, Cav1.3 subunit ( Cacna1d gene) immunoprecipitation. ( B ) SK3 subunit ( Kcnn3 gene) immunoprecipitation. ( C ) Left panel, HCN2 subunit immunoprecipitation. Right panel, HCN4 subunit immunoprecipitation. ( D ) Kv4.3 ( Kcnd3 gene) immunoprecipitation. High-confidence Class A (solid line) and low-confidence Class B (dashed lines) thresholds are displayed on the plots. Plain squares represent the bait and empty square interactants. The most relevant of the significant interactants are named.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Affinity Purification, Mass Spectrometry, Immunoprecipitation

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Visualization of the mass-spectrometry-based protein interaction network. The 6 ion channels of interest are depicted using different colors (blue, Kv4.3; pink and red, HCN2 and HCN4; yellow and orange, Cav1.2 and Cav1.3; green, SK3). Interactions are shown with lines of according colors. Thick lines represent Class A interactions, thinner lines Class B. Putative interactants were compared with the CRAPome 2.0 database (Contaminant Repository for Affinity Purification). Proteins identified in less than 1% of all CRAPome negative experiments were considered highly specific (black circles), between 1 and 2%, specific (grey circles), and between 2 and 10%, weakly specific (white circles) interactants.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Mass Spectrometry, Affinity Purification

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Cav1.3-HCN2-SK3 complex characterization in SNc DA neurons using proximity ligation assay. ( A ) Left, representative images of the PLA reaction (green) in wild-type (Wt) or Cav1.3 −/− SNc DA neurons using anti-Cav1.3 and anti-HCN2 antibodies. Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( B ) Representative images of the PLA reaction (green) in wild-type (Wt) SNc DA neurons using anti-SK3 and anti-HCN2 antibodies (top row) or anti-SK2 and anti-HCN2 antibodies (bottom row). Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( C ) Quantification of PLA fluorescent puncta on SNc DA neurons. Left, Cav1.3 and HCN2 PLA from 3 Wt (empty black circles; n = 21 images) or 3 Cav1.3 −/− (empty red circles; n = 18 images) mice. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. Middle, HCN2 and SK3 PLA (empty black circles; n = 11 images) or HCN2 and SK2 (empty orange circles; n = 7 images) from 2 independent experiments or anti-Kv4.3 and HCN2 (empty grey circle; n = 4 images) PLA from 1 experiment. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. Right, Cav1.3 and SK3 PLA from 2 Wt (empty black circles; n = 10 images) or 2 Cav1.3 −/− (empty red circles; n = 8 images) mice. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. ** p < 0.005; *** p < 0.001, Wilcoxon–Mann–Whitney test.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Proximity Ligation Assay, Immunolabeling, MANN-WHITNEY, Immunofluorescence

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Characterization of the interactions between SK3 and Tsc1 in SNc DA neurons using proximity ligation assay. ( A ) Left, representative images of the PLA reaction (green) in SNc DA neurons using anti-Tsc1 and anti-SK3 antibodies (top row) or anti-Tsc1 and anti-SK2 antibodies (bottom row). Middle, immunolabeling of SNc DA neurons with anti-TH antibodies (red). Right, merged images. Scale bars, 10 μm. ( B ) Quantification of PLA fluorescent puncta in SNc DA neurons using anti-Tsc1 and SK3 antibodies (empty black circles; n = 16 images) or anti-Tsc1 and SK2 antibodies (empty orange circles; n = 14 images) from 2 independent experiments. Superimposed box and whiskers plots allow us to visualize the median and interquartile range. ** p < 0.005, Wilcoxon–Mann–Whitney test.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Proximity Ligation Assay, Immunolabeling, MANN-WHITNEY, Immunofluorescence

Journal: Cells

Article Title: High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

doi: 10.3390/cells13110944

Figure Lengend Snippet: Schematic representation summarizing the ion channel interactions detected with co-immunoprecipitation, mass spectrometry and proximity ligation assay. Plain lines indicate co-immunoprecipitated ion channels. Dashed lines indicate interactions detected in mass spectrometry experiments. The arrows point from the bait to the prey. Dotted lines link proteins detected in close proximity using PLA in situ in SNc DA neurons.

Article Snippet: Affinity purified antibodies against Cav1.2, Cav1.3, HCN2, HCN4, Kv4.3, and SK3 subunits were obtained from Alomone in BSA-free buffer.

Techniques: Co-Immunoprecipitation Assay, Mass Spectrometry, Proximity Ligation Assay, Immunoprecipitation, In Situ, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Western Blot