iodophenpropit  (Alomone Labs)

Journal: PLoS ONE

Article Title: Early Proliferation Does Not Prevent the Loss of Oligodendrocyte Progenitor Cells during the Chronic Phase of Secondary Degeneration in a CNS White Matter Tract

doi: 10.1371/journal.pone.0065710

Figure Lengend Snippet: Myelin internode length following partial ON transection. Representative images of a single slice from the z stacks show ventral axons of control animals (A) and at 3 months following injury (B) anterogradely traced with CTB (green); paranodes are immunohistochemically labelled with Caspr and nodes with Nav1.6. C: The length of myelin internodes (indicated by

Article Snippet: Animals were transcardially perfused 3 days following CTB injection, ONs were post-fixed and longitudinal sections (14 µm) incubated overnight at 4°C with primary antibodies mouse anti-Caspr (1∶750, Abcam) and rabbit anti-Nav1.6 (1∶125, Alamone Laboratories) secondary antibody AlexaFluor® 546 or 647 and sections mounted with ProLong® Gold.

Techniques: CtB Assay

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Pharmacological inhibition of Kir7.1 in vitro in human and murine myometrium induces membrane depolarization and long-lasting contractions Representative membrane potential recording in current clamp configuration from a murine GD15 myometrial strip. (i) Addition of 10 μM VU590 depolarizes resting membrane potential (note slope change from resting potentials of preceding phasic bursts) and leads to a sustained plateau potential. (ii) Upon washout, spike potentials recover as plateau potential hyperpolarizes. (iii) On complete washout, phasic bursting resumes. Addition of 1 nM oxytocin and 10 μM VU590 to human myometrial strips stimulates long-lasting contractions. (i) Initial component of the response is phasic, followed by establishment of a tonic contraction.

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: Inhibition, In Vitro, Stripping Membranes

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Knockdown of Kir7.1 in vivo significantly increases intrauterine pressure Mice in which Kir7.1 was knocked down (Anti-Kir7.1) had significantly increased intrauterine pressure when compared to mice injected with scrambled miRNA lentivirus (scrambled) from GD13 to GD18 (Fig 6 and Supplementary Fig S3 ). ( n = 6 per time point). * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: In Vivo, Mouse Assay, Injection

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Pharmacological inhibition of Kir7.1 in vitro in human and murine myometrium stimulates longer-lasting contractions than oxytocin Dose- and gestation-dependent effect of VU590 on murine myometrial contractility [ n = 5, activity integral expressed as a fold-change of pre-treatment contractions (mean ± SD, per GD)]. * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: Inhibition, In Vitro, Activity Assay

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Model of the role of Kir7.1 in maintenance of uterine quiescence In normal labour in the human myometrium, changes take place over a number of weeks that increase both the intrinsic electrical excitability of the cell (blue box) and, by altering cellular receptors and contractile machinery, the susceptibility to stimulation. Alterations in stimulation either by infection or gene-environment interaction can precipitate preterm labour (Cha et al , 2013 ); however, changes in control of the myometrial membrane potential or interference with functional gap junctions can affect labour even under normal endocrine conditions (Bond et al , 2000 ; Doring et al , 2006 ). In this study, we demonstrate that alteration of the function of a single potassium channel profoundly alters uterine contractility. Loss of Kir7.1 function depolarizes the plasma membrane and promotes voltage-gated calcium entry. In addition, the duration of the depolarization is extended, preventing the normal phasic contractions. In this way, the process of excitation-contraction coupling in uterine myocytes overrides pharmaco-contraction coupling. This effect could be used for therapeutic benefit. (Key: CPI-17 = C-kinase potentiated protein phosphatase-1 inhibitor. CX26 = Connexin 26. CX43 = Connexin 43. CX45 = Connexin 45. GEFs = Guanine nucleotide exchange factors. GPCR = G-protein coupled receptors. IP3 = inositol 1,4,5-trisphosphate. IP3R = inositol 1,4,5-trisphosphate receptor. MLC = Myosin light chain. MLCp = Phosphorylated myosin light chain. PIP2 = Phosphatidylinositol 4,5-bisphosphate. PLC β = Phospholipase C. RHOgtp = Ras homologue gene family, member A. RHO-K = Rho-associated, coiled-coil containing protein kinase 1.)

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: Infection, Functional Assay, Planar Chromatography

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Stimulation of uterine contractions correlates with Kir7.1 inhibitory potency A The structures of the three compounds tested in this study. BNBI, a potent Kir1.1 inhibitor does not inhibit Kir7.1. MRT2000769 is structurally unrelated to VU590 and was identified as inhibiting Kir7.1 in a high throughput electrophysiology screen. VU590 is the first described inhibitor of Kir7.1. B, C Effects of BNBI (B) and MRT2000769 (C) on murine GD15 myometrial contractility.

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: High Throughput Screening Assay

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Kir7.1 is expressed in uterine myocytes and is regulated in pregnancy in mice and humans mRNA expression of Kcnj13 in mice ( n = 5; mean ± SD, per GD) normalized to GD13. * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: Mouse Assay, Expressing

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Knockdown of Kir7.1 in vitro depolarizes resting membrane potential A–C Representative membrane potential recordings in current clamp configuration from murine myometrial strips (A) treated with scrambled control miRNA (B) treated with Kir7.1 knockdown (Anti-Kir7.1) and (C) overexpressing Kir7.1 (+Kir7.1). D Resting membrane potential in current clamp configuration from murine myometrial strips ( n = 6; mean ± SD) from experiments depicted in (5A–C). * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: In Vitro

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Knockdown of Kir7.1 in vitro increases myometrial activity and promotes tonic contractions A–C Representative time-force recordings of phasic contractions demonstrating (A) the effect of scrambled miRNA compared to (B) knockdown (Anti-Kir7.1) and (C) overexpression (+Kir7.1) of Kir7.1 on contractility in murine GD15 myometrial strips. D–F Mean data are summarized ( n = 8; mean ± SD, per group of experiments) as activity integral (area under the time-force curve) (D), contraction duration (E) and maximum force (F). * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques: In Vitro, Activity Assay, Over Expression

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: A free-running simulation of the effect on the myometrial action potential waveform of increasing densities of Kir7.1 Time-dependent effect of increasing Kir7.1 channel densities on V m (mV, left) and [Ca] i (nM, right). Increasing density of Kir7.1 within experimentally determined values hyperpolarizes resting membrane potential, whereas decreasing membrane excitability during depolarizing excursions in V m leading to decreased calcium entry.

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques:

Journal: EMBO Molecular Medicine

Article Title: The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

doi: 10.15252/emmm.201403944

Figure Lengend Snippet: Kir7.1 current in uterine myocytes decreases from mid-pregnancy to term Measurement of inwardly rectifying, VU590-sensitive current in freshly dissociated GD15 murine myometrial cells. Shown are voltage-clamp recordings in the presence and absence of 10 μM VU590. Current-voltage relation ( n = 5; mean ± SD, per data point) of current density [VU590 subtracted from control (vehicle alone)]. Current density (pA/pF) at −150 mV and 500 ms in freshly dissociated murine myometrial cells from GD15 and GD18 ( n = 5 mean ± SD, per GD). * P

Article Snippet: The membrane was blocked in 5% milk protein solution (Marvel, Lincs, UK) for 1 h at room temperature, incubated with primary rabbit polyclonal anti Kir7.1 antibody (1:200; Alomone Labs, Jerusalem) overnight at 4°C in blocking buffer and then incubated with polyclonal goat anti-rabbit HRP secondary antibody (1:100; Dako, Ely, UK).

Techniques:

Journal: Brain Structure & Function

Article Title: A critical role for the inward rectifying potassium channel Kir7.1 in oligodendrocytes of the mouse optic nerve

doi: 10.1007/s00429-020-02043-4

Figure Lengend Snippet: Expression of Kir7.1 in optic nerve oligodendrocytes. a Optic nerve sections from PLP-DsRed reporter mice to identify oligodendrocytes (here shown in magenta), showing oligodendrocyte somata immunopositive for Kir7.1 (some indicated by asterisks), while there is less immunostaining in the myelinated fascicles ( ai , overlay image; aii , aiii , individual channels; ai inset, negative control pre-incubated in blocking peptide. b Oligodendrocytes from optic nerve explant cultures immunostained for Kir7.1; bi illustrates the overlay, where co-labelling appears white; bii and biii are the individual channels for Kir7.1 and the PLP-DsRed reporter respectively. c Colocalisation analysis of Kir7.1 and PLP-DsRed in situ in the P15 mouse optic nerve (white indicates voxels in which the magenta and green channels are expressed at the same level). d Mean thresholded Pearson’s correlation coefficient (PCC), showing significantly greater colocalisation between Kir7.1 and PLP-DsRed in oligodendroglial somata than in myelin (* p

Article Snippet: Incubation in rabbit anti-Kir7.1 antibody at 1:200 (Alomone) was carried out overnight at 4 °C, and following washes, the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Agilent; Santa Clara, CA, USA) was added at 1:5000 for 2 h at RT; controls were preincubated with the competitive peptide from which the Kir7.1 antibody was raised.

Techniques: Expressing, Plasmid Purification, Mouse Assay, Immunostaining, Negative Control, Incubation, Blocking Assay, In Situ, Periodic Counter-current Chromatography

Journal: Brain Structure & Function

Article Title: A critical role for the inward rectifying potassium channel Kir7.1 in oligodendrocytes of the mouse optic nerve

doi: 10.1007/s00429-020-02043-4

Figure Lengend Snippet: Kir7.1 channels are important for oligodendrocyte survival. Optic nerves from P12–P14 SOX10-eGFP mice were used to identify somata of oligodendrocyte lineage cells (OL), exposed for 1 h to normal oxygen and glucose (OGN) or oxygen and glucose deprivation (OGD) conditions, in the presence or absence of the specific Kir7.1 inhibitor VU590 (100 µM). a , b Representative images of optic nerves from SOX10-eGFP reporter mice incubated in OGN ( ai ) and OGD ( bi ) and in OGN + VU590 ( aii ) and OGD + VU590 ( bii ). c Quantification of the number of SOX10-eGFP positive cells (mean ± SEM; n = 4 nerves per group) revealed a dependence of OL cell survival on Kir7.1. It is also apparent that there is significant disruption and loss of OL cells during OGD compared to OGN, which was exacerbated by VU590 (*** p

Article Snippet: Incubation in rabbit anti-Kir7.1 antibody at 1:200 (Alomone) was carried out overnight at 4 °C, and following washes, the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Agilent; Santa Clara, CA, USA) was added at 1:5000 for 2 h at RT; controls were preincubated with the competitive peptide from which the Kir7.1 antibody was raised.

Techniques: Mouse Assay, Incubation

Journal: Brain Structure & Function

Article Title: A critical role for the inward rectifying potassium channel Kir7.1 in oligodendrocytes of the mouse optic nerve

doi: 10.1007/s00429-020-02043-4

Figure Lengend Snippet: Kir7.1 mediate potassium currents in optic nerve oligodendrocytes. Whole-cell patch clamp analysis in oligodendrocytes from optic nerve explant cultures. a VU590-sensitive Kir7.1-like currents. 10 mV depolarising voltage steps from − 130 to + 60 mV were applied in high extracellular K + in the absence of any pharmacological agents ( ai ), in the presence of the specific Kir7.1 blocker VU590 (20 µM) ( aii ) or VU590 and the Kir4.1 blocker BaCl 2 (100 µM) ( aiii ). b, c The results ( n = 4) are plotted as I–V relations before and after exposure to Kir blockers ( b ), and peak current density is expressed relative to the no drug condition in the same cells, illustrating a decrease in the presence of the blockers at − 130 mV ( ci ) as well as at + 60 mV ( cii ) (*** p ≤ 0.001, **** p ≤ 0.0001; one-way ANOVA with Tukey’s multiple comparison’s test, n ≥ 4)

Article Snippet: Incubation in rabbit anti-Kir7.1 antibody at 1:200 (Alomone) was carried out overnight at 4 °C, and following washes, the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Agilent; Santa Clara, CA, USA) was added at 1:5000 for 2 h at RT; controls were preincubated with the competitive peptide from which the Kir7.1 antibody was raised.

Techniques: Patch Clamp

Journal: Brain Structure & Function

Article Title: A critical role for the inward rectifying potassium channel Kir7.1 in oligodendrocytes of the mouse optic nerve

doi: 10.1007/s00429-020-02043-4

Figure Lengend Snippet: Kir7.1 expression in the mouse optic nerve. a qRT-PCR analysis of Kir channels in acutely isolated optic nerves from adolescent (P9–12) and young adult (P30–40) mice; * p

Article Snippet: Incubation in rabbit anti-Kir7.1 antibody at 1:200 (Alomone) was carried out overnight at 4 °C, and following washes, the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Agilent; Santa Clara, CA, USA) was added at 1:5000 for 2 h at RT; controls were preincubated with the competitive peptide from which the Kir7.1 antibody was raised.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Mouse Assay

Journal: Brain Structure & Function

Article Title: A critical role for the inward rectifying potassium channel Kir7.1 in oligodendrocytes of the mouse optic nerve

doi: 10.1007/s00429-020-02043-4

Figure Lengend Snippet: Model of Kir7.1 function in oligodendrocytes. Axonal action potential propagation results in continuous K + release into the extracellular space, which is taken up by oligodendrocytes through Kir4.1 and by the activity of Na + –K + pumps. Potassium is redistributed through Kir7.1, which acts to protect the cell in the face of these depolarizing influences and to recycle K + that is essential for maintaining Na + –K + pump activity. Blockade of Kir7.1 with VU590 results in oligodendrocyte demise and this is exacerbated in ischaemia, where Na + –K + pumps are compromised

Article Snippet: Incubation in rabbit anti-Kir7.1 antibody at 1:200 (Alomone) was carried out overnight at 4 °C, and following washes, the secondary antibody horseradish peroxidase-conjugated goat anti-rabbit (Agilent; Santa Clara, CA, USA) was added at 1:5000 for 2 h at RT; controls were preincubated with the competitive peptide from which the Kir7.1 antibody was raised.

Techniques: Activity Assay

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: Kir7.1 channel blocker VU590 inhibits c-wave originating from RPE cell. ( A ) Representation of the scotopic traces at different intensities from control mice and mice injected with VU590 or VU-608, an inactive analogue of VU-590. ( B ) Amplitude of a-wave and ( C ) b-wave after VU590 injection (red) compared with the VU608 injected eye (blue) and the control (black) saline injected eye. ( D ) Scotopic ERG trace representing the reduction of c-wave after the injection of VU590 but not with VU608 when compare with control. ( E ) Graphical representation of the c-wave reduction after Kir7.1 channel inhibition. Data is mean ± SEM and *P

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques: Mouse Assay, Injection, Inhibition

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: Kir7.1 protein expression. ( A ) Protein expression of Kir7.1 in RPE and retinal tissue detected by Western blot analysis. Complete Western blot image is included as Supplemental Fig. 2A . ( B ) Bar graph showing the relative expression of Kir7.1 protein in the RPE and the retina by densitometry expressed as a Kir7.1/b-actin ratio. ( C ) Immunohistochemistry against Kir7.1 and Ezrin; ezrin (red) labels the microvilli of the RPE cells and Kir7.1 (green) co-localizes with ezrin confirming its presence in the apical processes of the RPE cell. Outer nuclear layer (ONL) is stained with DAPI (blue). Scale bar is indicated. A video is included as a Supplemental data. ( D ) Higher magnification image of the RPE layer shows ezrin expression (red) in apical processes extending towards the retina. Kir7.1 (green) staining also appeared in apical membrane extensions with co-localization of both proteins (yellow) in long apical processes (arrow). Scale bar is included. ( E ) A quantitative distribution plot representation of signals acquired in green (488) vs. red (594).

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: Control of sub-retinal space K + homeostasis by Kir7.1 is crucial for photoreception. On the left is a representation of a normally functioning Kir7.1 channel which is able to maintain normal K + levels. On the right, a reduction in K + in the sub-retinal space due to disease that alters the ERG. The model: 1- Depolarization of RPE, 2- Changes in the sub-retinal space volume and K + , and 3- PR ionic conductance. RPE: retinal pigment epithelium, PR: photoreceptor, R SRS : sub-retinal space resistance.

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques:

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: Reduced RPE/retina function after sub-retinal administration of lentivirus containing shRNA for Kir7.1. ( A ) Gel electrophoresis showing that Kir7.1 mRNA expression is reduced after Kir7.1 shRNA injection when compared to the un-injected contralateral eye. Full image of the gel is included as Supplemental Fig. 3A . (B ) Expression of Kir7.1/b-actin ratio in shRNA injected and un-injected eye (part A) represented in a bar graph. ( C ) Representative scotopic traces comparing the shRNA-injected mice 14 d post injection, as well as control and PBS-injected mice. ( D ) Fluorescent image of live RPE sheet of cells displaying the expression of GFP that is fused with the shRNA Kir7.1. ( E ) Normalized a-wave and ( F ) normalized b-wave comparing the shRNA injected eye with the PBS injected eye at 30 cd.s/m 2 at 0, 2, 4, 7, and 14 d post-injection. ( G ) Comparison of c-wave for eyes injected with Kir7.1 shRNA and PBS at 0, 2, 4, 7 and 14 d post injection. Dotted line represents the control-normalized response from non-injected eyes.

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques: shRNA, Nucleic Acid Electrophoresis, Expressing, Injection, Mouse Assay

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: mRNA expression of Kir7.1 in RPE and retina. ( A ) Gel electrophoresis image illustrates the expression of Kir7.1 mRNA in both RPE and retinal tissue, as well as in pools of 10 isolated cells. Complete gel image is included as Supplemental Fig. 1A . ( B ) Images of representative single RPE, bipolar, and Müller glial cell used for single-cell RNA extraction. ( C ) mRNA expression for Kir7.1 and GAPDH in RPE cells (R1–R3), bipolar cells (B1–B3), and Müller glial cells (M1-M2) along with negative control water and perfusion bath solution. A Supplemental Fig. 1C is included showing full gel image.

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Nucleic Acid Electrophoresis, Isolation, RNA Extraction, Negative Control

Journal: Scientific Reports

Article Title: Abnormal Electroretinogram after Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

doi: 10.1038/s41598-017-11034-1

Figure Lengend Snippet: Inhibition of Kir7.1 current by VU590. Representative traces of whole cell currents recorded from single isolated RPE cells from mouse ( A ) and CHO-K1 cells ( B ) overexpressing the Kir7.1 channel, respectively. Whole cell currents were recorded by voltage ramping from +40 mV to −160 mV. The traces represent current density, which is the whole cell current normalized for the cell capacitance. In both cell types, the normal baseline Kir7.1 current is recorded prior to cells being treated with 50 µM VU590. The current is reduced after treatment with VU590, but is not affected by the treatment with 50 µM VU608. ( C ) and ( D ) The normalized Kir7.1 current at −160 mV is compared before, and after, treatment with VU590 or VU608.

Article Snippet: The frozen sections were rehydrated with PBS × 10 min and incubated with blocking solution containing 5% goat serum, 2% BSA and 0.3% Triton X-100 for 1 h. Primary antibodies were prepared in a 1:3 dilution of blocking solution containing antibody specific to Kir7.1 pre-labelled with ATTO-488 (1:50, Alomone labs, Jerusalem, Israel) and ezrin (1:200, Cell Signaling Technology, Danvers, MA).

Techniques: Inhibition, Isolation