Journal: BMC Medicine

Article Title: Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

doi: 10.1186/1741-7015-2-30

Figure Lengend Snippet: Primers

Article Snippet: Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)].

Techniques:

Journal: BMC Medicine

Article Title: Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

doi: 10.1186/1741-7015-2-30

Figure Lengend Snippet: Quantification of KCNJ10 and KCNQ1 mRNA expression in stria vascularis and spiral ganglia of Slc26a4 +/+ and Slc26a4 -/- mice. a: Electropherogram of total RNA isolated from stria vascularis microdissected from one mouse. The amount of total RNA was obtained from the total integral ( shaded ) and the amount of 18S rRNA was obtained from the integral of the 18S peak. Sharp peaks representing 18S and 28S rRNA demonstrate the high quality of RNA. Insert: Genotype of Slc26a4 -/- mice was verified by the observation of one or few very large rhomboedric otoconia in the utricular macula ( arrow ). A, crista ampullaris; U, utricular macula. Scale bar: 100 μm. b: Example of real-time RT-PCR data used for quantification of 18S, KCNJ10, KCNQ1 and KCNQ4. Known quantities of 18S rRNA were used to calibrate the threshold. SV, stria vascularis; SG, spiral ganglia. c: Quantification of KCNJ10 and KCNQ1 mRNA in young Slc26a4 +/+ and young and old Slc26a4 -/- mice.

Article Snippet: Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)].

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: BMC Medicine

Article Title: Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

doi: 10.1186/1741-7015-2-30

Figure Lengend Snippet: Protein localization of KCNJ10 in the cochlea of Slc26a4 +/+ and Slc26a4 -/- mice. a: Overview of cochlea; bar = 100 μm. Compare to Fig. 1a to note the enlarged scala media and the distended Reissner's membrane. b-c: Detail of lateral wall and spiral ganglia ( insert ); main bar: 10 μm, insert: 5 μm. Expression of KCNJ10 in Slc26a4 -/- mice was absent in stria vascularis but unchanged in spiral ganglion cells. RM, Reissner's membrane, SV, stria vascularis; SP, spiral prominence; SL, spiral ligament; LIM, spiral limbus; SG, spiral ganglion.

Article Snippet: Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)].

Techniques: Expressing

Journal: BMC Medicine

Article Title: Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model

doi: 10.1186/1741-7015-2-30

Figure Lengend Snippet: Model for the loss of KCNJ10 in the absence of pendrin expression in stria vascularis. Cys, cysteine, Glu, glutamate, Gly, glycine, CA, carbonic anhydrase, GST, glutathione-S-transferase, GSH, glutathione.

Article Snippet: Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)].

Techniques: Expressing

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: bioRxiv

Article Title: Characterization of TMEM43 as a novel ion channel

doi: 10.1101/2022.11.08.515259

Figure Lengend Snippet: (A) Representative whole-cell I-V curves from naïve CHO-K1 cell and TMEM43 -expressing cell before (pink) and after (black) BaCl 2 treatment (100 μM shown). (B) Summary bar graph of currents plotted at -150 mV (I -150mV ) and +50 mV (I +50mV ) for each condition in (A). (C) Co-immunoprecipitation results of TMEM43 expressed with TASK-1 and K ir 4.1. Cell lysates were immuno-pulled with an anti-TMEM43 antibody and blotted with TASK-1 and K ir 4.1 antibodies. TASK-1 was shown as a positive control.

Article Snippet: Bound proteins were eluted from the beads with SDS-PAGE sample buffer, and western blotting was performed with mouse anti-TASK-1(KCNK3) (1:250, NBP2-42202, Novus) or rabbit anti-K ir 4.1 (1:250, APC-035, Alomone Labs).

Techniques: Expressing, Immunoprecipitation, Positive Control

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: A–D LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p‐GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML‐SI1 treatment. (A) Representative images of CA1 pyramidal neurons stained with anti‐p‐GluA1 S845 (magenta), anti‐GluA1 (red), and anti‐GFP (green) antibodies. Scale bar = 20 µm. (B, C, D) Quantitative analysis of the mean fluorescence intensity (MFI) of p‐GluA1 S845 (B), GluA1 (C)‐immunoreactivity, and the ratio of p‐GluA1 to GluA1 (D) in hippocampal CA1 stratum radiatum (SR) 30 min after LFS. N = 4–6 slices from 4 to 6 mice. E Representative images of proximal dendrites of hippocampal neurons stained with anti‐GluA1 (green), anti‐LAMP2 (red), and anti‐GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false‐colored green and GFP false‐colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly colocalized puncta. Scale bar, 5 µm. F Quantitative analysis of the ratio of the number of GluA1/LAMP2 colocalized puncta to that of total lysosomes in (F) ( n = 17–29 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. * P < 0.05, ** P < 0.01 compared with shSc, # P < 0.05, ## P < 0.01 compared with shLAMTOR1; two‐way ANOVA with Tukey’s post‐test (B, C, D, F). A.U., Arbitrary unit. See also Appendix Fig . Source data are available online for this figure.

Article Snippet: Rows were successively loaded with antibody diluent, anti‐TRPML1 (1:10, Alomone) or anti‐LAMTOR1 (1:10, CST), horseradish peroxidase–conjugated anti‐rabbit secondary antibody, and a luminol‐peroxide mix.

Techniques: Staining, Fluorescence, Labeling

Journal: The EMBO Journal

Article Title: LAMTOR1 inhibition of TRPML1‐dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function

doi: 10.15252/embj.2021108119

Figure Lengend Snippet: Antibodies, chemicals, and plasmids used in this study.

Article Snippet: Rows were successively loaded with antibody diluent, anti‐TRPML1 (1:10, Alomone) or anti‐LAMTOR1 (1:10, CST), horseradish peroxidase–conjugated anti‐rabbit secondary antibody, and a luminol‐peroxide mix.

Techniques: Recombinant, In Situ, CRISPR, Plasmid Preparation

Journal: eLife

Article Title: Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit

doi: 10.7554/eLife.71379

Figure Lengend Snippet:

Article Snippet: Antibody , Kir 4.1 extracellular (rabbit polyclonal) , Alomone Labs , APC-165 , Immunofluorescence (1:200).

Techniques: Sequencing, Western Blot, Immunofluorescence, Transduction

Journal: bioRxiv

Article Title: Compartment-Specific Neurexin Nanodomains Orchestrate Tripartite Synapse Assembly

doi: 10.1101/2020.08.21.262097

Figure Lengend Snippet: Ultrastructural data on excitatory synapses after astrocyte-specific deletion of Nrxn1, extending the data shown in Fig. 3g & 3h ( a, postsynaptic spine area; b, PSD thickness; c, PSD length; d, number of docked synaptic vesicles/synapse; e, number of synaptic vesicles per terminal; f, width of synaptic cleft). g-l, Immunoblotting analyses show that the neuron- ( g, i, k ) or astrocyte-specific ( h, j, l ) Nrxn1 deletions do not produce detectable changes in the levels of selected synaptic proteins ( g, h ), neurexin ligands ( i, j ), or astrocytic proteins ( k, l ) in the hippocampus analyzed at P26-P30. Representative images of immunoblots (left of each column) and summary graphs of protein levels (right of each column; normalized to controls) are shown. Proteins are organized into groups as indicated. Numerical data are means ± SEM. n = indicated above except, 6-8 ctrl / 6-8 neuron cKO; 8 ctrl / 8 astrocyte cKO, except Glt1 and Kir4.1 with 4/4 and 3/3, respectively ( g-l ).

Article Snippet: The following antibodies were used at the indicated concentrations (IHC-immunohistochemistry; ICC-immunocytochemistry; IB-immunoblot): purified anti-HA mouse (Biolegend Cat# 901501; 1:500 live surface ICC, 1:500 ICC, 1:500 IHC, 1:1000 IB), purified anti-HA mouse Alexa647-conjugated (Biolegend Cat# 682404; 1:500 IHC), anti-HA rabbit (Cell Signaling Cat#3724; 1:250 ICC), anti-Homer1 rabbit (Millipore Cat# ABN37; 1:1000 IHC) anti-Nrxn1 rabbit (Synaptic Systems Cat# 175103; 1:1000 IB), anti-pan-Nrxn rabbit (Frontier Institute Cat# AF870; 1:500 IB), anti-pan-Nrxn rabbit (homemade, G393; 1:500 IB), anti-pan-Nrxn rabbit (homemade, G394; 1:500 IB), anti-pan-Nrxn rabbit (Millipore Cat# ABN-161-l; 1:1000 IB), anti-laminin-alpha2 rat (Abcam Cat# ab11576; 1:5000 IHC), anti-GFAP mouse (Neuromab Cat# 75-240; 1:1000 IB, 1:1000 ICC, 1:500 IHC), anti-GFAP rabbit (Agilent Cat# 033401-2; 1:1000 ICC, 1:1000 IHC), anti-GFAP chicken (Encorbio Cat# CPCA-GFAP; 1:1000 ICC, 1:1000 IHC), anti-HS-stub 3G10 antibody mouse (Amsbio Cat# 370260-1; 1:1000 IB), anti-vGluT1 (Homemade YZ6089; 1:1000 IHC, 1:1000 IB), anti-MAP2 mouse (Sigma Cat# M1406; 1:1000 IHC), anti-NeuN mouse (Millipore Cat# MAB377; 1:1000 IHC), anti-NeuN rabbit (1:1000 IHC), anti-ß-actin mouse (Sigma Cat#A1978; 1:3000 IB), anti-Synapsins rabbit (Homemade YZ6078; 1:500 ICC, 1:1000 IB), anti-Flag rat (Sigma Cat# SAB4200071; 1:500 surface ICC), anti-Myc rat (Abcam Cat# ab206486; 1:500 surface ICC), anti-VGAT guinea pig (Synaptic Systems Cat# 131005; 1:500 IHC), anti-GluN1 mouse (Synaptic Systems Cat# 114011; 1:1000 IB), anti-GluN2B mouse (Neuromab Cat# 75-101; 1:1000 IB), anti-GluR1 rabbit (Millipore Cat# Ab1504; 1:1000 IB), anti-GluR2 mouse (Neuromab Cat# 75-002; 1:1000 IB), anti-GluR4 (Millipore Cat# Ab1508; 1:1000 IB), anti-PSD-95 mouse (Neuromab Cat# 75-028; 1:1000 IB), anti-GRIP mouse (1:1000 IB), anti-Gad67 mouse (Millipore Cat# mab5406B; 1:1000 IB), anti-SNAP25 rabbit (Homemade P913; 1:500 IB), anti-Nlgn1 mouse (Synaptic Systems Cat# 129111; 1:1000 IB), anti-Nlgn2 rabbit (Synaptic Systems Cat# 129203; 1:1000 IB), anti-Nlgn3 mouse (Synaptic Systems Cat# 129311; 1:2000 IB), anti-GluD1 rabbit (Frontier Institute Cat# GluD1C-Rb-Af1390; 1:2000 IB), anti-CASK mouse (NeuroMab Cat# 75-000; 1:1000 IB). anti-Kir4.1 rabbit (Alomone Cat# APC-035; 1:2000 IB), anti-Glt1 guinea pig (Millipore Cat# AB1783; 1:1000 IB)

Techniques: Western Blot