rabbit polyclonal anti-slc12a2  (Millipore)

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: (A) Thirty-second trace of an Slc12a2 K842 * /K842 * and control mouse. Turns: blue, CC; red, C; green, none. (B) The majority of mutant mice have a bias in the C or CC direction. Slc12a2 K842 * /K842 *, C: n = 22; CC: n = 17; NP: n = 3. Foxg1 Cre/+ ; Slc12a2 fx/fx , C: n = 11; CC: n = 10. BAC 316.23, C: n = 3; CC: n = 5; NP: n = 1. Pax2-Cre;Tbx1 fx/fx , C: n = 4; mean ± SEM. See also . (C) The directional preference is stable over time. Mean percent of clockwise circles: Slc12a2 K842 * /K842 * C turners, day 1 versus 5, p > 0.9999; CC turners, day 1 versus 5, p > 0.9999; Foxg1 Cre/+ ; Slc12a2 fx/fx C turners, day 1 versus 31, p > 0.9999; CC turners, day 1 versus 31, p = 0.42. Mean ± SEM, repeated measures ANOVA with Bonferroni multiple comparison test. See also . BAC, bacterial artificial chromosome; C, clockwise; CC, counterclockwise; NP, no preference; SEM, standard error of the mean.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques: Control, Mutagenesis, Comparison

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: (A) Illustration of the contralateral and ipsilateral hemispheres regarding turning direction. (B) fEPSPs were greater ( p = 0.017) on the ipsilateral compared to contralateral side in Slc12a2 K842 * /K842 * mice. Inset: standard cortico-striatal fEPSP recording (black) and the sensitivity of the EPSP component to NBQX, an AMPA receptor antagonist (red); NP1: presynaptic fiber volley. fEPSPs between left and right hemispheres of littermate controls ( p = 0.94) or of mutants ( p = 0.96). Numbers in parentheses: number of recordings, number of mice. Mean ± SEM, two-way repeated measures ANOVA. See also . AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; C, clockwise; CC, counterclockwise; EPSP, excitatory postsynaptic potential; fEPSP, field excitatory postsynaptic potential; NP1, negative peak 1; SEM, standard error of the mean.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques:

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: Western blot analysis and quantification indicate that ERK phosphorylation is up-regulated in the striatal hemisphere contralateral to the preferred direction of signaling in Foxg1 Cre/+ ; Slc12a2 fx/fx (contralateral versus ipsilateral, p = 0.027, n = 6 for mutants and n = 6 for controls) and BAC 316.23 mutants (contralateral versus ipsilateral, p = 0.045, n = 5 for mutants and n = 3 for controls). No significant differences in unphosphorylated ERK were detected between hemispheres in all comparisons. Mean ± SEM, two-tailed t-test. See also . BAC, bacterial artificial chromosome; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; SEM, standard error of the mean.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques: Western Blot, Phospho-proteomics, Two Tailed Test

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: (A) Illustration of the hypothesized dependence of the preferred turning direction on asymmetric striatal p-ERK levels and how SL327 inhibition could reduce or reverse the directional preference; C, CC. (B) SLC327-inhibition of p-ERK in the contralateral ventral striatum and vehicle in the ipsilateral side of Slc12a2 K842 * /K842 * mutants reduces clockwise circling 1 day post injection ( n = 4; p = 0.0007 with two-tailed Chi-square and Fisher exact tests), while bilateral vehicle has no effect ( n = 7). SL327 injected ipsilaterally trended toward increasing the already high clockwise circling 1 day post injection ( n = 7). Repeated measures ANOVA with the Tukey multiple comparison test; mean ± SEM. See also . (C) PD0325901 inhibition of p-ERK in the contralateral ventral striatum and vehicle in the ipsilateral side of Slc12a2 K842 * /K842 * mutants also reduces clockwise circling 1 day post injection ( n = 8; p < 0.0001 with two-tailed Chi-square and Fisher exact tests), while bilateral vehicle has no effect ( n = 8). PD0325901 injected ipsilaterally, as with SL327, trended toward increasing the already high clockwise circling 1 day post injection ( n = 8). Repeated measures ANOVA with the Tukey multiple comparison test; mean ± SEM. See also . C, clockwise; CC, counterclockwise; ERK, extracellular signal-regulated kinase; MEK, mitogen activated protein kinase kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; SEM, standard error of the mean.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques: Inhibition, Injection, Two Tailed Test, Comparison

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: (A) Immunohistochemical staining of inner ear sections showed reduced SLC12A2 expression (brown) in Tbx1 Cre/+ ; Slc12a2 fx/fx mutants (Nissl counterstain, purple) and defects of the cochlea (top panel) and vestibular structures: saccular vestibular membrane collapse (middle) and ut and cr degeneration (bottom). (B) ABR testing revealed no waveforms at 100 decibels, sound pressure level, in 6-week-old mutants, indicative of deafness. (C) VsEP (P1, first peak; P2, second peak) were absent in mutants, indicative of vestibular impairment. (D) Mutants performed poorly on the rotarod test ( p < 0.0001), consistent with impaired balance. Mean ± SEM, n = 8 mice/genotype, two-tailed t test. See also . (E) Open field recordings of Tbx1 Cre/+ ; Slc12a2 fx/fx mutants showed a turning bias in either the C ( n = 8) or CC ( n = 10) direction (NP, n = 1). Mean ± SEM. See also . (F) Tbx1 Cre/+ ; Slc12a2 fx/fx mutants circle ( n = 37), whereas littermate controls ( n = 10) and Nestin-Cre;Slc12a2 fx/fx mutants do not ( n = 5). Mesp1 Cre/+ ; Slc12a2 fx/fx mutants, in which Slc12a2 expression is lost from the brain vasculature (Antoine et al., 2017), also do not circle. Mean ± SEM. See also . (G) Western blots of striatal lysates from Tbx1 Cre/+ ; Slc12a2 fx/fx mutants showed greater p-ERK1 in the contralateral relative to ipsilateral hemisphere, normalized to β-actin ( p = 0.046, n = 6). Asymmetries in p-ERK1 levels were not detected in Nestin-Cre;Slc12a2 fx/fx mutants ( n = 3). Mean ± SEM, two-tailed t test. See also . ABR, auditory brain stem response; C, clockwise; CC, counterclockwise; cr, cristae; NP, no preference; p-ERK, phosphorylated extracellular signal-regulated kinase; rm, Reissner’s membrane; SEM, standard error of the mean; SPL, sound pressure level; sv, stria vascularis; ut, utricle; vm, vestibular membrane; VsEP, vestibular sensory evoked potential.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques: Immunohistochemical staining, Staining, Expressing, Membrane, Two Tailed Test, Western Blot

Journal: PLoS Biology

Article Title: Early uneven ear input induces long-lasting differences in left–right motor function

doi: 10.1371/journal.pbio.2002988

Figure Lengend Snippet: (A) Open field recordings of Tbx1 Cre/+ ; Slc12a2 fx/fx mutants ( n = 10) before (day −1) and after unilateral surgery on the left ear showed a preference for CC turning by 14 days. See also . (B) Average CC and C turns for the mutants in panel A and littermate controls ( n = 10) with unilateral ear surgery. Two-way RM ANOVA with Tukey’s multiple comparison test (between mutant CC and C: at day 14, p = 0.0051; day 21, p = 0.0019; day 25, p < 0.0001). See also . (C) Activation during caloric vestibular stimulation of the right and left ear in a right- and left-hander with numbers of voxels exhibiting significant differences in effect size between left and right hemispheres (paired t test; n = 12 right-handers, n = 12 left-handers; p < = 0.001). See also . (D) Model of ear-induced lateralization. C, clockwise; CC, counterclockwise; fEPSP, field excitatory postsynaptic potential; p-ERK, phosphorylated extracellular signal-regulated kinase; RM, repeated measure.

Article Snippet: Primary antibodies include mouse NeuN (1:100, Millipore, Burlington, MA), rabbit GFAP (1:500, Dako, Santa Clara, CA), goat SLC12A2 (1:50, Santa Cruz Biotechnology, Dallas, TX), rabbit SLC12A2 (1;100, Proteintech Group, Rosemont, IL), rabbit Laminin (1:200, Millipore), rabbit GFP (1:100, Life Technologies, Carlsbad, CA), rabbit p-ERK (1:200, Cell Signaling, Danvers, MA), mouse Islet1 (1:2, Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse Pou3f1/Oct6 (1:50, Millipore).

Techniques: Comparison, Mutagenesis, Activation Assay

Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: Representative confocal microscope images show extent of expression and subcellular localization of TMEM16A, SLC26A4, carbonic anhydrase 2 (CA2), SLC12A2, and ATP12A (images taken from BE37 cells; similar results were obtained from BE63 cells). Whenever permitted by the combination of primary antibodies, acetylated tubulin and MUC5AC were also stained as markers of ciliated and goblet cells, respectively. Bronchial epithelia were kept under control conditions or treated with IL-4 for 72 hrs. Larger images: xy sections (scale bar: 20 μm). Inset: xz sections (scale bar: 10 μm). Images with a different scale of view are shown in .

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques: Microscopy, Expressing, Staining

Journal: Scientific Reports

Article Title: Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

doi: 10.1038/srep36016

Figure Lengend Snippet: For simplicity, the cartoon shows all channels and transporters within the same cell although some components (e.g. CFTR and TMEM16A) are localized in separate cell types. The NKCC1 transporter (SLC12A2) promotes the intracellular accumulation of Cl − that is then secreted through TMEM16A and CFTR Cl − channels. Bicarbonate is accumulated inside the cell by means of basolateral transporters and by conversion from CO 2 . Pendrin then mediates the exchange of extracellular Cl − with intracellular HCO 3 − . The apical membrane also contains the ATP12A K + /H + pump and possibly a K + channel. Secretion of K + could be the mechanism controlling the acidification of apical fluid by ATP12A.

Article Snippet: The following primary antibodies and dilutions were used: rabbit monoclonal anti-TMEM16A [SP31] (ab64085, Abcam) at 1:200, mouse IgG1 anti-CFTR (ab570, J.R. Riordan, University of North Carolina at Chapel Hill, and Cystic Fibrosis Foundation Therapeutics) at 1:250, mouse polyclonal anti-SLC26A4 (H00005172-A01, Abnova) at 1:200, rabbit polyclonal anti-SLC12A2 (HPA020130, Sigma-Aldrich) at 1:1000, rabbit monoclonal anti-CA2 [EPR5195] (ab124687, Abcam) at 1:500, mouse IgG1 anti-MUC5AC (NCL-HGM-45M1, Novocastra) at 1:100, mouse IgG2B anti-acetylated tubulin (7451, Sigma Aldrich) at 1:300, rabbit polyclonal anti-ATP12A (HPA039526, Sigma-Aldrich) at 1:400.

Techniques: