Journal: Communications Biology

Article Title: A maladaptive feedback mechanism between the extracellular matrix and cytoskeleton contributes to hypertrophic cardiomyopathy pathophysiology

doi: 10.1038/s42003-022-04278-9

Figure Lengend Snippet: Immunoblot analysis of L-type calcium channel ( a , b ) and β 1 integrin ( c , d ) protein expression performed on total heart homogenate pooled from groups of 5 pre- (10–15-wk-old) or post-cardiomyopathic (30–50-week-old) cTnI-G203S mice and age-matched wt counterparts. Representative immunoblots probed with L-type calcium channel α 1C subunit (Ca V 1.2, a ) or β 1 integrin ( c ) antibody, then GAPDH monoclonal antibody. Densitometry analysis of Ca V 1.2 ( b ) or β 1 integrin ( d ) protein expression, normalized to associated GAPDH expression. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( b ) or Kruskal-Wallis test ( d ) determined statistical significance. Densitometry analysis of relative mTOR expression (calculated as Phospho-mTOR/Total mTOR) performed on cytoplasmic ( e ) and nuclear ( f ) fractions pooled from groups of five pre- or post-cardiomyopathic cTnI-G203S mice and age-matched wt counterparts. β-tubulin and histone H2B antibodies were used as loading controls for cytoplasmic and nuclear fractions respectively. n = number of technical repeats. A Browne-Forsythe and Welch ANOVA ( e ) or Kruskal-Wallis test ( f ) determined statistical significance. g Schematic indicating structural-functional link between the L-type calcium channel, cytoskeletal network, mitochondria, integrin and the extracellular matrix in wt and cTnI-G203S cardiac myocytes. A disrupted cytoskeletal architecture in cTnI-G203S cardiac myocytes may trigger a maladaptive feedback mechanism between increased cytoskeletal and extracellular matrix stiffness, resulting in a hypermetabolic mitochondrial state.

Article Snippet: Blots were probed with the following primary antibodies: rabbit polyclonal anti-Ca V 1.2 (Alomone, ACC-003, 1:200) or rabbit monoclonal anti-β 1 integrin (D6S1W) (Cell Signaling Technology 34971, 1:1000).

Techniques: Western Blot, Expressing, Functional Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing the ACAT1 Gene in Human SH-SY5Y Neuroblastoma Cells Inhibits the Expression of Cyclo-Oxygenase 2 (COX2) and Reduces β-Amyloid-Induced Toxicity Due to Activation of Protein Kinase C (PKC) and ERK

doi: 10.12659/MSM.912862

Figure Lengend Snippet: Primary antibodies used in the present study.

Article Snippet: Rabbit anti-Cacna1b , 1: 1000 , Alomone Labs, Jerusalem, Israel.

Techniques:

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing the ACAT1 Gene in Human SH-SY5Y Neuroblastoma Cells Inhibits the Expression of Cyclo-Oxygenase 2 (COX2) and Reduces β-Amyloid-Induced Toxicity Due to Activation of Protein Kinase C (PKC) and ERK

doi: 10.12659/MSM.912862

Figure Lengend Snippet: Effects of knockdown ACAT1 on CACNAs, PKC, and ERK phosphorylation and in Aβ-induced SH-SY5Y cells. ( A ) Western blot results showed that the relative expression of CACNA1b and CACNA2d1 was increased significantly, while the total PKC and the ratio of phospho-ERK (p-ERK)/ERK were decreased significantly after Aβ 25-35 treatment. ( B ) Knockdown of ACAT1 rescued the reduction of PKC and phosphorylation level of ERK, and also retarded the upregulation of CACNA1b and CACNA2d1 induced by Aβ. (** P <0.01 and * P <0.05).

Article Snippet: Rabbit anti-Cacna1b , 1: 1000 , Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Representative current traces recorded under control conditions (upper left panel) and in the presence of the specific β 2 AR agonist salmeterol alone (10 µM; left middle panel) or in combination with PKI 14–22 amide (10 µM; lower left panel). The bar graph represents D inact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with four cells recorded under 10 µM salmeterol and five cells recorded under 10 µM salmeterol plus PKI 14–22 amide. Data are presented as means ± SEM of several independent experiments. *** P <0.001. Significance of salmeterol plus PKI (n = 5) versus salmeterol alone (n = 4) was calculated by Student's t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. ( B ) Close co-expression of the main modulator of CDI, PKA (green) and Ca V 1.2 (red) in cultured neurons. Yellow dots represent places were these two proteins are in close proximity. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted without signal (negative control). ( C ) Indicated brain regions were immunostained with antibodies specific for PKARIIβ and Ca V 1.2. Thalamic regions LGN and VB revealed very strong interaction patterns in merged pictures. Association of these proteins is still present in hippocampus but on lower level. DG (dentate gyrus), PoDG (polymorph layer of the dentate gyrus).

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Expressing, Cell Culture, Negative Control

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Immunocytochemical analysis of primary cultures of the dorsal thalamus using Ca V 1.2- (red), AKAP150- (green) and PKARIIβ- (blue) specific antibodies. Merged picture shows the close connection of the components of the proposed ternary complex, especially in somatic regions and proximal dendrites. Enlarged inlay represents a magnification of the area indicated by the rectangle. Data shown are representative pictures from several independent immunostainings and preparations of neurons. In all cases, omission of primary antibodies resulted without signal (negative control). Western blot analysis and pull down assays were done as described in " ". ( B ) Interaction of AKAP7-MBP and PKARIIβ-c-myc was detected using antibodies against c-myc. ( C ) IP of PKARIIβ-c-myc and AKAP5-GFP detected after incubation with GFP-coupled magnetic beads using antibodies derived against PKARIIβ. ( D ) Existence of PKA holoenzyme consisting of PKARIIβ-GST and PKAcsβ-GFP was detected with antibodies against GFP protein.

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Negative Control, Western Blot, Incubation, Magnetic Beads, Derivative Assay

Journal: PLoS ONE

Article Title: Modulation of Calcium-Dependent Inactivation of L-Type Ca 2+ Channels via β-Adrenergic Signaling in Thalamocortical Relay Neurons

doi: 10.1371/journal.pone.0027474

Figure Lengend Snippet: ( A ) Representative current traces recorded under control conditions (upper left panel) and in the presence of okadaic acid (OA) (10 µM; left down panel). The bar graph (right panel) represents D inact under different recording conditions (as indicated). The mean value of three cells recorded under control conditions was taken for comparison with three cells recorded under 10 µM OA. ( B ) Representative current traces recorded under control conditions (upper left panel) and in the presence of the isoproterenol alone (10 µM; left middle panel) or in combination with OA (10 µM; lower left panel). The bar graph (right panel) represents D inact under different recording conditions (as indicated). The mean value of five cells recorded under control conditions was taken for comparison with five cells recorded under 10 µM isoproterenol and five cells recorded under isoproterenol plus OA. Data are presented as means ± SEM of several independent experiments. *** P <0.001. Significance of isoproterenol plus OA (n = 5) versus controls (n = 5) was calculated by Student's t test. ** P <0.01. Significance of isoproterenol (n = 5) versus isoproterenol plus OA (n = 5) was calculated by Student's t test. The degree of inactivation is given by the normalized current amplitude of the mean postpulse I/V at +10 mV. ( C ) Immunocytochemical analysis of primary cultures of the dorsal thalamus using Ca V 1.2- (left panel, green) and PP2A-specific antibodies (right panel, red). Merge picture showed close association of the two proteins, especially in somatic regions and proximal dendrites. Data shown are representative pictures from several independent immunostainings and thalamic neurons preparations. In all cases, omission of primary antibodies resulted in no fluorescence signal above background (negative control).

Article Snippet: After 1 h, the following primary antibodies were added in different combinations to the blocking solution and incubated for 90 min at room temperature: rabbit anti-Ca V 1.2 (1∶200, Alomone Labs, Israel); mouse anti-cAMP-dependent protein kinase type II beta regulatory subunit (PKARIIβ, 1∶500, BD Bioscience, USA); rabbit anti-β 2 -AR (H-73 1∶400, Santa Cruz, USA); mouse anti-microtubule-associated protein 2 (MAP2, HM-2, 1∶1000, Sigma, Germany); goat anti-AKAP-150 (N-19, Santa Cruz, USA); sheep anti-protein phosphatase 2A (PP2A, Acris, Germany).

Techniques: Fluorescence, Negative Control

Journal: Neural Development

Article Title: Temporal-spatial changes in Sonic Hedgehog expression and signaling reveal different potentials of ventral mesencephalic progenitors to populate distinct ventral midbrain nuclei

doi: 10.1186/1749-8104-6-29

Figure Lengend Snippet: Cells labeled with Shh-GIFM at different time points have a changing potential to contribute to subpopulations of DA neurons . (A-H) Immunofluorescent staining for β-gal-positive fate-mapped cells (Shh-GIFM at E8.5 or E11.5) and DA neurons (TH) on coronal sections of the ventral midbrain (P21 to P30). The areas shown in (A-D) are indicated in (E-H). Arrows indicate double-labeled cells; arrowheads indicate β-gal-positive cells with astrocytic morphology. (E'-H') Representative schematics of the immunostained sections showing the distribution of TH-positive fate-mapped cells (red dots) and of fate-mapped cells with astrocytic morphology (yellow crosses). Rostral, Bregma -2.92; caudal, Bregma -3.40 . If, interfascicular nucleus; Pn, paranigral nucleus; Snc, substantia nigra pars compacta; Snl, substantia nigra lateralis; Vta, VTA. Fate-mapped cells outside these areas are not represented. Cells with astrocytic morphology are not present with TM8.5. Scale bars: (A-D) 40 μm; (E-H) 200 μm. (I) Relative contribution of cells marked with Shh-GIFM between E8.5 and E11.5 to the SN (Snl + Snc), dorsal-lateral VTA (Vta) and ventral-medial VTA (Pn + If); see schematic in (L). For each animal (n ≥ 3), TH-positive fate-mapped cells were counted in the three indicated areas and normalized for the combined number of overlapping cells counted in these areas (in percent). Error bars indicate standard deviation. Significance (* P < 0.05; ** P < 0.01; *** P < 0.001) was determined by ANOVA and LSD post-hoc analysis. (J) Distribution of Calbindin- and Girk2-positive cells. (K) Relative contribution of fate-mapped cells to Calbindin (VTA) versus Girk2 (SN) positive cells. Calbindin- or Girk2-positive fate-mapped cells were counted in three different rostral-caudal midbrain areas (n ≥ 3). The ratio of Calbindin-positive fate-mapped cells to Girk2-positive fate-mapped cells was determined. Significance (*** P < 0.001) was determined by Student's t -test. (L) Schematic showing the SN, dlVTA and vmVTA. (M) Fate mapping strategy.

Article Snippet: Primary antibodies: goat anti-β-galactosidase (β-gal; 1:2,000, AbD Serotec, Oxford, UK), rabbit or rat anti-GFP (1:400, Invitrogen, Carlsbad, CA, USA or 1:2,000, Nacalai, Kyoto, Japan) rabbit or mouse anti-tyrosine hydroxylase (TH; 1:500, Millipore, Billerica, MA, USA), rabbit anti-Calbindin (1:5,000, Swant, Marly, Switzerland), rabbit anti-Lmx1a (1:2,000, Millipore), mouse anti-Pou4f1 (1:100, Santa Cruz Antibodies, Santa Cruz, CA, USA), rabbit anti-glial fibrillary acidic protein (GFAP; 1:500, Millipore), mouse anti-glutamine synthetase (1:500, Millipore) and rabbit anti-Girk2 (1:100, Alomone Labs, Jerusalem, Israel).

Techniques: Labeling, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

doi: 10.1371/journal.pone.0006586

Figure Lengend Snippet: A, Immunocytochemistry experiments show colocalization (overlay, yellow) of KCNQ2 (red) and syntaxin 1A ( syx ; green) in rat hippocampal neurons. High colocalization areas of KCNQ2 and syntaxin 1A are indicated by arrows. B, Colocalization of KCNQ2, syntaxin 1A and VAMP-2 in rat hippocampal neurons as detected by triple immunocytochemistry and illustrated by the merge images. KCNQ2 (red), syntaxin 1A (green) and VAMP-2 (blue) are indicated in the top images from left to right. The bottom images from left to right show the colocalization of KCNQ2 and VAMP-2 (merge, pink), syntaxin 1A and VAMP-2 (merge, light blue) and KCNQ2 and syntaxin 1A (merge, yellow). A varicosity colocalized with VAMP-2, syntaxin 1A and KCNQ2 is indicated by arrow. C, The same image as in B showing all three markers; KCNQ2 (red), syntaxin 1A (green) and VAMP-2 (blue). A linescan was placed through the varicosity indicated by arrow in B. The varicosity was shown to colocalize all three signals and thus, is indeed a synaptic one.

Article Snippet: Neurons were incubated at 4°C overnight with two or more of the following primary antibodies diluted in PBS containing 3% HS: a goat polyclonal antibody to KCNQ2 (N19, 1∶150; Santa Cruz Biotechnology Inc., Santa Cruz, CA), a mouse monoclonal anti-syntaxin 1A (1∶2500; Sigma-Aldrich, St. Louis, MO) and a rabbit polyclonal anti-VAMP-2 (Synaptobrevin 2; 1∶800, Alomone Laboratories, Jerusalem, Israel).

Techniques: Immunocytochemistry

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: Primary Antibodies Used

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: NK1R expression is localized to enteric neuron cell bodies and varicosities whereas NK2R is expressed primarily by neuronal varicosities surrounding enteric glia. Images (confocal single optical planes, 1 μm) show triple-label immunohistochemistry for GFAP (green, A-D), Hu (blue, A’–D’), and either NK1R (magenta, A’’-B’’) or NK2R (magenta, C’’–D’’) in the distal colon myenteric plexus of mice. Preadsorption control is shown for NK1R (B–B’’’’) and NK2R (D–D’’’’). Images show triple-label immunohistochemistry for GFAP (green, E), tdTomato-tagged TRPV1-positive varicosities (blue, E’), and NK2R (magenta, E’’) in the distal colon myenteric plexus of mice. ( A’’’–E’’’ ) Overlays of each combination. Areas demarked by the dashed boxes in A’’’–E’’’ are enlarged in panels A’’’’–E’’’’ . ( E’’’ ) Scale bar : 20 μm (applies to A–E’’’ ). Scale bars , 10 μm in the enlarged images (A’’’’, B’’’’, C’’’’, D’’’’, and E’’’’). Labeling is representative of experiments performed on a minimum of 3 mice.

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Expressing, Immunohistochemistry, Labeling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: Glial Ca 2+ responses driven by NKA require the activation of NK2Rs and purinergic intercellular signaling mediated by Cx43. ( A–D ) Representative glial Ca 2+ responses evoked by NKA in the presence of ( A ) tetrodotoxin, ( B ) the NK1R antagonist CP 96345, ( C ) the NK2R antagonist GR 159897, or ( D ) the P2Y1R antagonist MRS 2500. Gray traces show the responses of individual glial cells, and the averaged response of all glia within the ganglion is shown in the green trace . ( E and F ) Representative glial Ca 2+ responses evoked by NKA in tissues from SOX10 ::CreER T2+/- Cx43 f/f mice in the ( E ) absence or ( F ) presence of GR 159897. ( G and H ) Quantification of the effects of CP 96345, GR 159897, tetrodotoxin, MRS 2500, or ablation of glial Cx43 in SOX10 ::CreER T2+/- Cx43 f/f mice on the ( G ) number and ( H ) length of response of glial Ca 2+ responses induced by NKA (n = 15–33 glia from 3 to 5 mice; 1-way analysis of variance; ** P < .01; ∗∗∗ P < .001; **** P < .0001). TTX, tetrodotoxin.

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: Effects of the NK2R antagonist GR 159897 on colonic inflammation and TAC1 mRNA levels during acute colitis and recovery in mice. ( A ) Schematic representation of the timeline of treatments during colitis. ( B ) Weight loss pattern after induction of DNBS colitis in mice (n = 5–16 mice; 2-way analysis of variance with multiple comparisons; ∗∗∗ P < .001; **** P < .0001). Note that inflamed mice treated with GR 159897 recover normal body weight sooner than inflamed mice treated with vehicle. ( C ) GR 159897 did not modify macroscopic damage to the colon at 48 hours or 3 weeks after induction of DNBS colitis in mice (n = 5–16 mice; 2-way analysis of variance with multiple comparisons; **** P < .0001). ( D ) GR 159897 did not modify histology scoring to the colon at 24 hours after induction of DNBS colitis in mice (n = 3–4 mice; 2-way analysis of variance with multiple comparisons; **** P < .0001). ( E ) TAC1 mRNA levels (normalized to glyceraldehyde-3-phosphate dehydrogenase [ GAPDH ]) in the distal colon myenteric plexus of healthy (vehicle) and inflamed (DNBS) mice treated with vehicle or GR 159897 at 48 hours or 3 weeks after induction of DNBS colitis (n = 4–7 mice; 2-way analysis of variance with multiple comparisons; * P = .0368).

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: The NK2R antagonist GR 159897 limits neuropathy and reactive gliosis in the myenteric plexus during inflammation. ( A ) Representative confocal microscopy images of dual-label immunohistochemistry for GFAP (green) and the pan-neuronal marker Hu (magenta) in healthy (saline) and inflamed (DNBS) mice treated with vehicle or GR 159897 at 48 hours or 3 weeks after induction of colitis. Scale bar : 30 μm (applies to all images). Labeling is representative of experiments performed on a minimum of 4 mice. ( B ) Quantification of myenteric neuron packing density in healthy and inflamed mice treated with vehicle or GR 159897 at 48 hours or 3 weeks after induction of colitis (n = 3–8 mice; 2-way analysis of variance with multiple comparisons; * P = .02239; ** P = .0097). ( C and D ) Quantification of ( C ) GFAP protein expression (integrated density, n = 4–6 mice; 2-way analysis of variance with multiple comparisons, normalized to group vehicle saline) and ( D ) GFAP mRNA levels (n = 4–7 mice; 2-way analysis of variance with multiple comparisons, normalized to glyceraldehyde-3-phosphate dehydrogenase [ GAPDH ]; * P = .0114) in samples of myenteric plexus from the distal colons of healthy and inflamed mice treated with vehicle or GR 159897 at 48 hours or 3 weeks after induction of colitis. ( E and H ) Quantification of glial morphology including ( E ) total process length, ( F ) process thickness, and Scholl analysis for process branching at ( G ) 48 hours or ( H ) 3 weeks after induction of colitis (n = 16 glia; 2-way analysis of variance with multiple comparisons; * P = .0185; *** P = .0003; **** P < .0001).

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Confocal Microscopy, Immunohistochemistry, Marker, Labeling, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: NKA-driven enteric neurodegeneration requires the activation of NK2Rs and Cx43 hemichannels. ( A ) Quantification and ( B ) representative images of the mean packing density of HuC/D-immunoreactive neurons in myenteric ganglia after in situ activation of P2X7Rs with BzATP or activation of NK2Rs with NKA in the presence or absence of the Cx43 inhibitor 43Gap26, the NK2R antagonist GR 159897, the P2X7 antagonist A74003, or the pannexin-1 inhibitor probenecid (n = 3 mice; 1-way analysis of variance with multiple comparisons; * P = .0157; ** P = .0073). Enteric neurons are labeled with HuC/D (magenta) and enteric glia are labeled with GFAP (green) in all panels. Scale bar : 20 μm (applies to all images).

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Activation Assay, In Situ, Labeling

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: Glial transcriptome alteration driven by acute inflammation. ( A ) Schematic of transgene containing the HA-tagged ribosomal protein L22 (Rpl22) driven by the tamoxifen inducible glial promoter (SOX10 creERT2 ). ( A’ ) Confocal image of dual-label immunohistochemistry (single optical plane, 1 μm) for the HA-tagged Rpl22 (magenta) and enteric glia (GFAP, green) in the myenteric plexus of the Sox10 ::CreER T2+/- / Rpl22 tm1.1Psam /J mouse distal colon. ( A'iii ) The area marked is enlarged in panel A'iv . ( A'iii ) Scale bar : 20 μm (applies to A'i–iii ). ( A'iv ) Scale bar : 10 μm. ( B ) Mean fragments per kilobase of transcript per million mapped reads (FPKM) values of sentinel glial and neuronal genes from vehicle saline samples showing increased relative expression of glial to neuronal transcripts (n = 3 mice; data shown represent biological replicates). ( C ) Total numbers of differentially expressed enteric glial genes in saline mice treated with the NK2R antagonist GR 159897 or vehicle (GR 159897 saline compared with vehicle saline; n = 3 mice; P < .005; data shown represent biological replicates). ( C’ ) Total numbers and ( C” ) Venn diagram comparison of differentially expressed enteric glial genes 48 hours after induction of DNBS colitis in mice treated with the NK2R antagonist GR 159897 or vehicle compared with vehicle saline mice. ( C–C” ) Up-regulated genes are shown in red and down-regulated genes are shown in blue. ( D ) Heatmaps for all 316 differentially expressed genes in the vehicle DNBS vs vehicle saline comparison in order of fold-change (positive to negative) for all groups (n = 3 mice; P < .005; data shown represent biological replicates). Color scale is based on gene-based Z-scores of log 2 FPKM values. ( E ) Selected differentially expressed genes classified by inflammation-related gene ontology (GO) terms from the distal colon of vehicle DNBS mice compared with vehicle saline mice (n = 3 mice; P < .005; data shown represent biological replicates). Superscripts denote actual categorization in similar, more specific GO terms, which were grouped together under a broader category of GO terms, as follows: 1 adaptive immune response; 2 innate immune response in mucosa; 3 antigen processing and presentation of peptide antigen via major histocompatibility complex class I; 4 negative regulation of antigen processing and presentation of peptide antigen via major histocompatibility complex class I; and 5 negative regulation of peptide or polysaccharide antigen via major histocompatibility complex class II. ( F ) Selected differentially expressed genes classified by biological process GO categories, which are significantly differentially regulated between GR 159897 DNBS and vehicle DNBS mice (n = 3 mice; P < .005; data shown represent biological replicates). Relative changes shown are compared with vehicle saline.

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Immunohistochemistry, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: NK2R antagonism prevents changes in neuromuscular transmission after colitis. ( A–D ) Neurogenic ( A and B ) contractions and ( C and D ) relaxations driven by EFS (20 V, 0.3 ms, 5 Hz) in colons from mice treated with vehicle or GR 159897 at 7 days after induction of colitis. ( A and B ) DNBS enhanced neurogenic contractions, ( C and D ) but did not significantly alter neurogenic relaxations. ( A and B ) Treatment with GR 159897 diminished the effects of DNBS colitis on neurogenic contractions (n = 3–4; 2-way analysis of variance with multiple comparisons; * P < .05; ** P < .01).

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Transmission Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Communication Between Enteric Neurons, Glia, and Nociceptors Underlies the Effects of Tachykinins on Neuroinflammation

doi: 10.1016/j.jcmgh.2018.05.009

Figure Lengend Snippet: Schematic model of the intercellular signaling mechanisms that underlie the effects of NKA on neuroinflammation in the ENS. NKA and SP released from intrinsic neuronal varicosities drive NK1R activation on enteric neurons and NK2R activation on nociceptive neurons and enteric neurons. Activation of NKRs drives adenosine triphosphate (ATP) release from enteric neurons that recruit activity in surrounding enteric glia by activating P2Y1Rs. Intracellular signaling pathways downstream of P2Y1R activation drive glial Ca 2+ responses that contribute to Cx43 hemichannel opening and ATP release that further enhances glial responses, drives P2X7R-mediated neuroinflammation, and P2Y1R activation on nociceptive neurons. ADP, adenosine diphosphate. eNTPdase, ectonucleoside triphosphate diphosphohydrolase 2.

Article Snippet: Rabbit anti-NK2R , Alomone Labs , 1:500 , ATR-002 , AB_2341078.

Techniques: Activation Assay, Activity Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Ca 2+ /Calmodulin-Dependent Protein Kinase II (CaMKII) Increases Small-Conductance Ca 2+ -Activated K + Current in Patients with Chronic Atrial Fibrillation

doi: 10.12659/MSM.909684

Figure Lengend Snippet: qRT-PCR and Western blotting results showing KCNN 1–3 mRNA levels and K Ca 2.1/2.2/2.3 proteins levels in the atria of SR controls (n=20) and AF patients (n=32). ( A ) mRNA levels of KCNN1, KCNN2, and KCNN3 in SR and AF. ( B ) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in SR group. ( C ) mRNA expression differences of KCNN1, KCNN2, and KCNN3 in AF group. ( D ) K Ca 2.1–2.3 (SK1–3) proteins expression changes in SR (n=20) and AF (n=32). * P <0.05 vs. SR.

Article Snippet: The PVDF membrane was incubated with rabbit polyclonal anti-KCa 2.1 (SK1), anti-KCa 2.2 (SK2), anti-KCa 2.3 (SK3, Alomone Labs, Jerusalem, Israel) (dilution 1: 500), rabbit polyclonal anti-CaM (Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1: 1000), rabbit polyclonal anti-CaMKII (Abcam, Cambridge, UK) (dilution 1: 1000), rabbit polyclonal anti-pCaMKII (Thr 286 , Cell signaling, USA) (dilution 1: 1000), anti-pCaMKII (Thr 287 , Abcam, Cambridge, UK) (dilution 1: 1000), and rabbit polyclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, USA) (dilution 1: 2000) overnight at 4ºC.

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Commercially Available Angiotensin II At 2 Receptor Antibodies Are Nonspecific

doi: 10.1371/journal.pone.0069234

Figure Lengend Snippet: Characteristics of AT 2 receptor antibodies used in the study.

Article Snippet: AT 2 , affinity purified rabbit pAb, Alomone Labs, cat # AAR-012 , Peptide DNLNATGTNESAFNC corresponding to amino acid residues 21-35 of rat AT 2 receptor , M, R , WB, IHC, ICC.

Techniques: Enzyme-linked Immunosorbent Assay, Affinity Purification

Journal: PLoS ONE

Article Title: Commercially Available Angiotensin II At 2 Receptor Antibodies Are Nonspecific

doi: 10.1371/journal.pone.0069234

Figure Lengend Snippet: Additional available AT 2 receptor antibodies not tested.

Article Snippet: AT 2 , affinity purified rabbit pAb, Alomone Labs, cat # AAR-012 , Peptide DNLNATGTNESAFNC corresponding to amino acid residues 21-35 of rat AT 2 receptor , M, R , WB, IHC, ICC.

Techniques: Affinity Purification, Enzyme-linked Immunosorbent Assay