Journal: International Journal of Molecular Sciences

Article Title: Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium

doi: 10.3390/ijms24021673

Figure Lengend Snippet: Immunolocalization of TRPV4 in the mouse ciliary body. TRPV4 (green) is abundant in the NPE (nonpigmented ciliary epithelium) cell layer at the surface but not the underlying pigmented ciliary epithelium (PE) cell layer or stroma. The lower panel shows a negative control in which no primary antibody was used.

Article Snippet: The knockout validated, rabbit polyclonal anti-TRPV4 antibody was purchased from Alomone Lab (Catalog # ACC-034; Jerusalem, Israel).

Techniques: Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium

doi: 10.3390/ijms24021673

Figure Lengend Snippet: The ATP release response to cyclic stretch was inhibited by the selective TRPV4 antagonists HC 067047 and RN-1734 but not by the TRPV1 antagonist A889425. The amount of ATP in the bathing medium was measured after subjecting monolayers of cultured NPE cells to 10% cyclic stretch (0.5 Hz) for 2 min in the presence of either HC 067047 (10 µM) ( A ) or RN-1734 (10 µM) ( B ) or A889425 (1 µM) ( C ) added 20 min beforehand. The data are the mean ± SE of results from six independent experiments. ** ( p < 0.01) and *** ( p < 0.001) indicate significant differences from control and ### ( p < 0.001) indicates significant differences from a stretch stimulus.

Article Snippet: The knockout validated, rabbit polyclonal anti-TRPV4 antibody was purchased from Alomone Lab (Catalog # ACC-034; Jerusalem, Israel).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium

doi: 10.3390/ijms24021673

Figure Lengend Snippet: The connexin mimetic peptide Gap 27 prevents ATP release and PI uptake responses to TRPV4 activation by a selective agonist, GSK1016790A. The amount of ATP in the bathing medium ( A ) or cell PI fluorescence ( B ) was measured after exposing the monolayers of cultured NPE cells to GSK1016790A (10 nM) in the presence or absence of Gap 27 (200 µM) added 60 min beforehand. The data are the mean ± SE of results from six independent experiments. *** ( p < 0.001) indicates significant differences from control.

Article Snippet: The knockout validated, rabbit polyclonal anti-TRPV4 antibody was purchased from Alomone Lab (Catalog # ACC-034; Jerusalem, Israel).

Techniques: Activation Assay, Fluorescence, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Mechanical Stretch Activates TRPV4 and Hemichannel Responses in the Nonpigmented Ciliary Epithelium

doi: 10.3390/ijms24021673

Figure Lengend Snippet: The TRPV4 antagonist HC067047 prevents ATP release and PI uptake responses to an osmotic swelling stimulus. The amount of ATP in the bathing medium ( A ) or cell PI fluorescence ( B ) was measured after exposing the monolayers of cultured NPE cells to hypoosmotic Kreb’s solution (200 mOsm) for 2 min in the presence or absence of HC067047 (10 µM) added 20 min beforehand. The data are the mean ± SE of results from twelve ( A ) or six ( B ) independent experiments. *** ( p < 0.001) indicates significant differences from control.

Article Snippet: The knockout validated, rabbit polyclonal anti-TRPV4 antibody was purchased from Alomone Lab (Catalog # ACC-034; Jerusalem, Israel).

Techniques: Fluorescence, Cell Culture

Journal: Frontiers in Physiology

Article Title: Impact of KvLQT1 potassium channel modulation on alveolar fluid homeostasis in an animal model of thiourea-induced lung edema

doi: 10.3389/fphys.2022.1069466

Figure Lengend Snippet: Impact of KvLQT1 activation on alveolar type I (AT1) and II (ATII) epithelial cell markers in thiourea-challenged WT mice. Representative immunofluorescence images (left panels) of lung sections (5 μm, Scale: 100 μm) from WT control mice (PBS/PBS), and WT mice challenged with thiourea (TU, 5 mg/kg i.p., PBS/TU) and treated or not (PBS) with the KvLQT1 activator R-L3 (R-L3/TU) stained with the ATI markers podoplanin (A) , n = 50 images, AQP5 (B) , n = 59–60 or ATII marker pro-SPC (C) , n = 48–51). Nuclei were stained by DAPI. Quantification (right panels) of podoplanin, AQP5, and pro-SPC marker intensities was made with a protocol exploited by ICY software. Values are presented as means ± SEM. Non-parametric Mann-Whitney t-test (Agostino/Pearson normality test: negative) for panel (A) . One-way ANOVA non-parametric comparison test (normality Agostino/Pearson test: negative) for panel (B , C) . * p < .05, ** p < .01 or **** p < .0001 vs . PBS/PBS, ## p < .01 vs . PBS/TU.

Article Snippet: For AQP5 detection by immunostaining, tissue sections were processed for heat-induced antigenic retrieval (with citrate buffer, pH 3.45) and then blocked with a solution of PBS +10% FBS (Saradigm, United States) + 10% BSA (Sigma-Aldrich) + .01% Triton X-100 (Amersham Biosciences, Sweden) for 1 h. Slides were then incubated overnight at 4°C with an anti-AQP5 rabbit polyclonal antibody (1:100, #AQP-005, Alomone Labs, Israël).

Techniques: Activation Assay, Immunofluorescence, Staining, Marker, Software, MANN-WHITNEY

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: (A) Experimental design for (B)–(H). (B) Representative images of maximal projection and 3D reconstruction (IMARIS) of mG + astrocytes. (C and D) Surface area and enclosed volume of mG + astrocytes. Graphed as means of each astrocyte in Aldh1l1-CreER T2 :Sox2 fl/fl mice (left, n = 28 control, 43 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (E–H) Automatic tracing of mG + astrocyte processes by the filament tool of IMARIS and quantifications. Graphed as means of each astrocyte (left, n = 19 control, 17 Sox2 icKO) and mouse (right, n = 3 control, 4 Sox2 icKO). (I) Left, experimental design for (J)–(P); right, immunofluorescence of SOX2 and GFAP and quantification. n = 4 control, 4 Sox2 icKO. (J) Ctx astrocytes visualized by SR101 (arrows) for whole-cell recordings. (K) Voltage steps for astrocyte recording, from −180 to +20 mV with a step size of 10 mV. (L and M) Representative current tracing (L) and I-V curve (M) of one Sox2 -deficient and one Sox2 -intact astrocyte. (N1–N3) Quantification of cell capacitance (N1, n = 23 control, 18 Sox2 icKO), input resistance (N2, n = 20 control, 19 Sox2 icKO), and resting membrane potential (N3, n = 20 control, 17 Sox2 icKO) of Ctx astrocytes. (O) Western blot and quantification of Kir4.1 in the brain. n = 4 control, 4 Sox2 icKO. (P) Representative images and quantification of Kir4.1 intensity in the cortex. n = 4 control, 4 Sox2 icKO. Error bars indicate means ± SEM. Unpaired two-tailed Student’s t test was used for statistically analyzing two groups of data. Please see for statistics. n, biological replicates. Scale bars, (B, E, I, J, and P) 20 μm.

Article Snippet: Rabbit polyclonal anti-Kir4.1 , Alomone labs , Cat# APC-035; RRID: AB_2040120.

Techniques: Immunofluorescence, Western Blot, Two Tailed Test

Journal: Cell reports

Article Title: SOX2 is essential for astrocyte maturation and its deletion leads to hyperactive behavior in mice

doi: 10.1016/j.celrep.2022.111842

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-Kir4.1 , Alomone labs , Cat# APC-035; RRID: AB_2040120.

Techniques: Protein Extraction, Recombinant, Electron Microscopy, Injection, Magnetic Cell Separation, Lysis, Bicinchoninic Acid Protein Assay, Western Blot, Glutamate Assay, Labeling, SYBR Green Assay, Chromatin Immunoprecipitation, Software, Activity Assay

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: Reproductive biology and endocrinology : RB&E

Article Title: Stimulation of TM3 Leydig cell proliferation via GABA A receptors: A new role for testicular GABA

doi: 10.1186/1477-7827-2-13

Figure Lengend Snippet: Sequences of oligonucleotide primers used for PCR amplification

Article Snippet: For immunohistochemistry, immunocytochemistry and Western blot analyses we employed rabbit polyclonal antiserum against GAD, which recognizes both isoforms GAD65 and GAD67 (DPC Biermann, Bad Nauheim, Germany); rabbit polyclonal antiserum against VGAT (SySy Synaptic Systems GmbH, Göttingen, Germany); rabbit polyclonal antiserum against GABA A -α1 (Alomone Labs Inc., Jerusalem, Israel), sheep polyclonal antiserum against GABA B -R1 and GABA B -R2 (gift from Graham Disney and Fiona Marshall, GlaxoWellcome R&D Inc., Stevenage, UK), mouse monoclonal antiserum against PCNA (Merck Biosciences, Schwalbach, Germany) and mouse monoclonal antiserum against β-Actin (Sigma, Deisenhofen, Germany).

Techniques:

Journal: Reproductive biology and endocrinology : RB&E

Article Title: Stimulation of TM3 Leydig cell proliferation via GABA A receptors: A new role for testicular GABA

doi: 10.1186/1477-7827-2-13

Figure Lengend Snippet: Leydig cells in postnatal rat testis possess GAD, VGAT and GABA A -α1. Immunohistochemical localization of GAD, VGAT and GABA A -α1 in the testis of five days old rats. Fetal Leydig cells with typical rounded morphology are located in clusters between the seminiferous tubules and are immunopositive for GAD (A) and VGAT (B). No reaction was observed in sections incubated with buffer (data not shown) or non-immune rabbit serum (C) instead of the primary antibody. The GABA A receptor subunit α1 (D) is also immunolocalized to clustered fetal Leydig cells (*). However other interstitial cells with a spindle-shaped appearance also exhibit specific immunoreactivity against GABA A -α1 (D, →). The insert panel (D) also depicts magnified spindle-shaped interstitial cells of another section, which are immunopositive for GABA A -α1. No reaction was observed in sections incubated with buffer (data not shown) or non-immune rabbit serum (E) instead of the primary antibody. Bars: 50 μm.

Article Snippet: For immunohistochemistry, immunocytochemistry and Western blot analyses we employed rabbit polyclonal antiserum against GAD, which recognizes both isoforms GAD65 and GAD67 (DPC Biermann, Bad Nauheim, Germany); rabbit polyclonal antiserum against VGAT (SySy Synaptic Systems GmbH, Göttingen, Germany); rabbit polyclonal antiserum against GABA A -α1 (Alomone Labs Inc., Jerusalem, Israel), sheep polyclonal antiserum against GABA B -R1 and GABA B -R2 (gift from Graham Disney and Fiona Marshall, GlaxoWellcome R&D Inc., Stevenage, UK), mouse monoclonal antiserum against PCNA (Merck Biosciences, Schwalbach, Germany) and mouse monoclonal antiserum against β-Actin (Sigma, Deisenhofen, Germany).

Techniques: Immunohistochemical staining, Incubation

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Cryostat sections of rat renal tissue were stained for TRPV1 by indirect immunofluorescence using polyclonal an antibody (left). Note that distinct tubular structures were positive for TRPV1 (arrows). Omission of the primary was used as negative control (right). B. At higher magnifications it was observed that PGP9.5 positive nerve fibers (arrows) within the rat kidney also express the capsaicin receptor TRPV1 as shown by the overlay of both.

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques: Staining, Immunofluorescence, Negative Control

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Increases in free intracellular calcium in response to repetitive application of NOD displayed marked tachyphylaxis from stimulus to stimulus (grey trace). Application of the competitive TRPV1 antagonist capsazepine before and during the 2 nd stimulus (CPZ; black trace) abolished the NOD response. B. Mean change in intracellular calcium by NOD in cells challenged with (filled bars; n = 4) and without CPZ during the second stimulus (open bars; n = 5) indicated the specificity of NOD at TRPV1. The threshold for a significant response is indicated by the dotted line (*p<0.05, ***p<0.001 vehicle versus CPZ, student’s unpaired t-test).

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques:

Journal: PLoS ONE

Article Title: N-octanoyl-Dopamine Is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Is Chemia-Induced Acute Kidney Injury in Rat

doi: 10.1371/journal.pone.0043525

Figure Lengend Snippet: A. Human PTECs were marked with DAPI and stained with a primary anti-TRPV1 antibody (giunea-pig polyclonal serum); omission of a primary antibody served as negative control ( B ). C. In addition TRPV1 was assessed in lysates of unstimulated (1) or TNF-α stimulated PTECs (2) by Western blotting. TRPV1 transfected HEK cells (3) were used as positive control to indicate the molecular weight of TRPV1.

Article Snippet: TRPV1 in frozen kidney sections was detected using rabbit anti-TRPV1 polyclonal primary antibody (Alomone) and secondary anti-rabbit Cy3 (Jackson ImmunoResearch, PA, USA).

Techniques: Staining, Negative Control, Western Blot, Transfection, Positive Control, Molecular Weight