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: 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: 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: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Bar graphs comparing the relative distributions of A 1 R(A1)- and P2Y 2 R(Y2)-immunoreactive elements in each brain region (A-C) and in transfected HEK293T cells (D) . The P2Y 2 R-P2Y 2 R, A 1 R-A 1 R and A 1 R-P2Y 2 R dimers are indicated by Y2-Y2, A1-A1 and A1-Y2, respectively. Total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Raw data are shown in the tables under the graphs. Data are means of three independent experiments.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-localization of A 1 R and P2Y 2 R . A-C . Confocal images of double immunostained Myc-P2Y 2 R (A; green), HA-A 1 R (B; red), and their merge (C; yellow) in co-transfected HEK293T cells. The co-localization of HA-A 1 R and Myc-P2Y 2 R is evident at the cell surface membrane (small arrow). D-L . Confocal images of double immunofluorescence staining in several rat brain regions. P2Y 2 R (D, G, J; red) and A 1 R (E, H, K; green) immunoreactivities were detected in Purkinje cells (D-F), cerebellar nuclei (G-I), and hippocampal CA3 pyramidal cells (J-L). Co-localizations of A 1 R and P2Y 2 R (F, I, L; yellow) were detected in the soma (large arrows) of all tissues, in dendrites of the Purkinje cells, and in neurons of the cerebellar nuclei (arrowheads). Yellow bar indicates 500 μm (A-C) and white bar indicates 100 μm (D-L). Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) each 10-μm optical slice consisted of a stack of 20 0.5-μm thick sections. Serial optical sections were recorded using an air objective lens of (40×, numerical aperture; 0.6).

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection, Double Immunofluorescence Staining, Confocal Laser Scanning Microscopy

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-immunoprecipitation of A 1 R from cerebral cortex (lane 1), cerebellum (lane 2), and hippocampus (lane 3) . Immunoblotting analyses of extracts of rat brain with anti-A 1 R (A), anti-P2Y 2 R (B), and anti-P2Y 2 R with the control peptide of P2Y 2 R (C). Membrane extracts from each region were immunoprecipitated with anti-A 1 R, and analyzed by immunoblotting with anti-P2Y 2 R (D), anti-P2Y 2 R with the control peptide of P2Y 2 R (E), anti-A 1 R (F). No bands were seen in C, E demonstrating the specificity of the antibodies. It was confirmed that immunoprecpitation without primary antibody resulted in no detectable receptor bands in the immunoblotting (data not shown). Approximate molecular masses are shown in kDa.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Immunoprecipitation, Western Blot

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Immunogold electron microscopy of A 1 R and P2Y 2 R visualized using nanogold particles in transfected HEK293T cells (A-D) and rat brain (E-G) . A: Localization of HA-A 1 R (large particles) detected with anti-HA in HA-A 1 R-transfected HEK293T cells. B: Localization of Myc-P2Y 2 R (small particles) detected with anti-Myc in Myc-P2Y 2 R-transfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of HA-A 1 R and Myc-P2Y 2 R in co-transfected HEK293T cells. D: HA-A 1 R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. E-G: Localization of A 1 R and P2Y 2 R in cortical pyramidal cells (E), Purkinje cells (F), and hippocampal pyramidal cells (G) detected with both anti-A 1 R and anti-P2Y 2 R. Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Electron Microscopy, Transfection, Incubation

Journal: Journal of Neuroinflammation

Article Title: Oral administration of the K ATP channel opener diazoxide ameliorates disease progression in a murine model of multiple sclerosis

doi: 10.1186/1742-2094-8-149

Figure Lengend Snippet: Western blotting show expression of both Kir6.1 and Kir6.2 K ATP channel pore-forming subunits in unstimulated and LPS/IFNγ-stimulated BV-2 cells (A, left) and microglial primary cultures (A, Right). Staining for the microglial cell membrane marker CD11b (B and E) and the K ATP channel subunits Kir 6.1 (C) or Kir 6.2 (F) showed colocalization in BV-2 microglia, indicating the expression of the K ATP channel at the cytoplasmic membrane (D and G, white arrows) . Control: unstimulated cells; L+I: cells stimulated with LPS and IFNγ. Scale bar = 30 μm.

Article Snippet: After washing in Tris-buffered saline (TBS: 20 mM Tris, 0.15 M NaCl, pH 7.5) for 5 min, dipping in methanol for 10 s and drying in air, the membranes were incubated with the following primary antibodies overnight at 4°C: polyclonal rabbit anti-Kir 6.1 or polyclonal rabbit anti-Kir 6.2 (both 1:500, Alomone, Jerusalem, Israel), polyclonal rabbit anti-inducible nitric oxide synthase (iNOS) (1:200, Millipore, Bedford, MA, USA), polyclonal rabbit anti-cyclooxygenase-2 (1:2000, Santa Cruz Biotechnology, St. Cruz, CA, USA) and monoclonal mouse anti-β-actin (1:50000, Sigma-Aldrich, St. Louis, MO, USA) diluted in immunoblot buffer (TBS containing 0.05% Tween-20 and 5% non-fat dry milk).

Techniques: Western Blot, Expressing, Staining, Marker