Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Ca 2+ -binding protein 2 inhibits Ca 2+ -channel inactivation in mouse inner hair cells

doi: 10.1073/pnas.1617533114

Figure Lengend Snippet: Normal number and gross molecular anatomy of IHCs and their ribbon synapses in Cabp2LacZ/LacZ mice. (A) Representative maximum projection of immunostaining for large-conductance Ca2+-activated K+ channels (BK, magenta) and parvalbumin-α (Pavalb., green) of mice at the age of P15. BK channels are clustered in the neck region of Cabp2LacZ/LacZ and Cabp2+/+ IHCs, indicating normal development. (Scale bars: 10 μm.) (B) Representative maximum projection of immunostaining for small-conductance Ca2+-activated K+ channels (SK2, magenta) and calretinin (Calret., green) of Cabp2LacZ/LacZ and Cabp2+/+ IHCs showing that SK2 puncta are restricted to OHCs and lacking in IHCs. The white arrowheads indicate SK2 channel puncta in OHCs. (Scale bars: 10 μm.) (C) Representative synapse immunostaining of Cabp2+/+ and Cabp2LacZ/LacZ IHCs of P21–P28 animals. Ribbon-occupied synapses were identified by colocalized presynaptic ribbon (RIBEYE/Ctbp2, magenta) and a postsynaptic bouton (GluA2/3, green). (Scale bars: 10 μm.) (D) Same experimental design as in C was also applied to organs of Corti of P56–P63 mice. (Scale bars: 10 μm.) (E, Left) Ribbon-occupied synapses are identified as juxtaposed spots of RIBEYE/Ctbp2 and GluA2/3 immunofluorescence (schematic with one exemplary synapse drawn). (E, Right) Quantification of ribbon-occupied synapses/IHC for Cabp2+/+ and Cabp2LacZ/LacZ IHCs of both age groups: No significant differences (n.s.) in synapse numbers were detected between genotypes of the respective age (3 wk: Wilcoxon rank sum test; 8 wk: Student’s t test). For all four conditions, organs of Corti of four animals were used. (F) Representative immunostaining of Ca2+ channels (CaV1.3, green) and the presynaptic ribbon (RIBEYE/Ctbp2, magenta) showing proper cluster formation of CaV1.3 at the active zone for Cabp2LacZ/LacZ and Cabp2+/+ IHCs. (Scale bars: 10 μm.)

Article Snippet: The following antibodies were used: guinea pig anti–parvalbumin-α (1:200; Synaptic Systems), rabbit anti-BK (1:100; Alomone), mouse anti–α-calbindin D-28k (1:400; SWANT), rabbit anti-SK2 (1:200; Alomone), mouse IgG1 anti-CtBP2 (1:200; BD Biosciences, rabbit anti-GluA2/3 (1:200;Chemicon), and rabbit–anti-Ca V 1.3 (1:100; Alomone).

Techniques: Immunostaining, Immunofluorescence

Journal: PLOS Pathogens

Article Title: Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

doi: 10.1371/journal.ppat.1011842

Figure Lengend Snippet: (A) Schematic representation of the regulation of S1P homeostasis by sphingosine kinase 1/2 (SphK1/2), S1P phosphatase 1/2 (SGPP1/2), S1P lyase (SGPL1) and S1P transporter Spinster 2 (Spns2). (B-G) Fold change in expression of SPHK1 (B), SPHK2 (C), SGPL1 (D), SGPP1 (E), SGPP2 (F) and SPNS2 (G) in hCMEC/D3s infected with N . meningitidis MC58 for an 8h infection time course normalized to 18S rRNA measured by qPCR. Data represent mean ± SD of 3 independent experiments measured in duplicates. Comparison to control for each time point using multiple t-test and p-value adjustment with Holm-Sidak correction. ***p<0.001 vs. respective control. (H) SphK enzymatic activity measured in hCMEC/D3s infected with N . meningitidis MC58 for a 6 h infection time in comparison to uninfected control cells as determined by ATP depletion assay. Uninfected control cells without addition of SphK substrate Sph (‘-Sph’) were used to detect non-specific ATP consumption. Graph represents the mean ± SD of at least three independent experiments with biological duplicates and is reported as activity normalized to control cells. One-Way ANOVA followed by by Dunett’s post-hoc test. *p<0.05, **p<0.01, ****p<0.0001 vs. control. (I) hCMEC/D3s were infected with N . meningitidis MC58 for 8h infection time course and both total protein and/or phosphorylated form of SphK1 (pSer-225) and SphK2 (pThr-578) were assessed by Western blot. Representative immunoblot is shown. Graphs represent the mean ± SD of three independent experiments and reported as phosphorylated form relative to total target protein normalized to control cells. One-Way ANOVA showed no significance. Related to .

Article Snippet: Rabbit polyclonal pospho-specific Anti-(Ser-225)-SphK1(ECM biosciences # SP1641) , 1:250 , 5% BSA in TBS-T.

Techniques: Expressing, Infection, Comparison, Activity Assay, ATP Depletion Assay, Western Blot

Journal: PLOS Pathogens

Article Title: Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

doi: 10.1371/journal.ppat.1011842

Figure Lengend Snippet: (A) hCMEC/D3 were co-transfected with SPHK1-siRNA (100 nM) and SPHK2-siRNA (25 nM) or transfected with scrambled siRNA (50 nM) for 24h and infected with N . meningitidis MC58 for 8h. Numbers of viable intracellular bacteria were determined by gentamicin protection assay and normalized to control cells (scrambled siRNA). Data are means ± SD; n = 4, N = 2. Unpaired students t-test, **** p < 0.0001. Absence of cytotoxicity and knockdown efficiency were verified as shown in . (B-D) hCMEC/D3 were treated with SphK1 inhibitor PF543 (B), SphK2 inhibitors SLM6031434 (C) or SphK2 inhibitor K145 (D) for 30 min or solvent control (DMSO) and subsequently infected with N . meningitidis MC58 for 8h. Numbers of viable intracellular bacteria were determined by gentamicin protection assay and normalized to control cells (DMSO treated cells). Data are means ± SD; n ≥ 4, N = 2. Comparison to DMSO-treated control using unpaired students t-test (A), multiple t-test with p-value adjustment with Holm-Sidak correction in comparison to control for each time point (B-D). ** p < 0.01, *** p < 0.001, **** p < 0.0001. Absence of cytotoxicity and bacteriostatic effects were verified as shown in .

Article Snippet: Rabbit polyclonal pospho-specific Anti-(Ser-225)-SphK1(ECM biosciences # SP1641) , 1:250 , 5% BSA in TBS-T.

Techniques: Transfection, Infection, Bacteria, Solvent, Comparison

Journal: PLOS Pathogens

Article Title: Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

doi: 10.1371/journal.ppat.1011842

Figure Lengend Snippet: Primers used for qPCR.

Article Snippet: Rabbit polyclonal pospho-specific Anti-(Ser-225)-SphK1(ECM biosciences # SP1641) , 1:250 , 5% BSA in TBS-T.

Techniques: Sequencing

Journal: PLOS Pathogens

Article Title: Sphingosine kinase 1/S1P receptor signaling axis is essential for cellular uptake of Neisseria meningitidis in brain endothelial cells

doi: 10.1371/journal.ppat.1011842

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Rabbit polyclonal pospho-specific Anti-(Ser-225)-SphK1(ECM biosciences # SP1641) , 1:250 , 5% BSA in TBS-T.

Techniques: Milk

Journal: bioRxiv

Article Title: Increased intrinsic membrane excitability is associated with hypertrophic olivary degeneration in spinocerebellar ataxia type 1

doi: 10.1101/2023.10.23.563657

Figure Lengend Snippet: (A) Transcriptomic analysis of whole medulla from SCA1-KI and wild-type mice at 28 weeks reveal that 31 ion channel genes are differentially expressed. Of these, 25 influence excitability intrinsically (IC intrinsic ), while 6 influence excitability synaptically (IC synaptic ). (B) Downregulation of Atxn1 expression by antisense oligonucleotides (ASOs) ameliorates brainstem phenotypes in SCA1-KI mice. Of the 1374 differentially expressed genes (DEGs) in the SCA1-KI medulla, 317 DEGs were significantly rescued by ASO treatment (and, therefore, are more likely to be responsible for the observed phenotypic rescue). Within this cohort, IC intrinsic genes were significantly enriched. (C) Heatmap showing differential expression of the 10 IC intrinsic DEGs between treatment groups. These ion channel genes exhibited a significant increase in expression after ASO treatment (left), with the majority of them rising to wild-type levels (right). (D) In order to examine how decreases in medullary ion channel transcripts may result in a loss of channels in the IOPr, immunostaining for the small-conductance potassium channel SK2 was performed. Coronal histological sections of the IOPr in wild-type (top) and SCA1-KI (bottom) mice at 14 weeks (an early symptomatic timepoint) are shown. Sections have been stained for SK2 (green) and calbindin (red). (E) Quantification of immunostaining reveals a significant loss of SK2 channels on the membrane of SCA1-KI IOPr neurons. Data are expressed as mean ± SEM. Statistical significance derived by Fischer’s exact test (enrichment analyses) or unpaired t -test with Welch’s correction (all other comparisons), ** = P < 0.01, ns = not significant. (A-C) N wild-type, Vehicle = 8 mice, N SCA1-KI, Vehicle = 8 mice, N SCA1-KI, ASO = 7 mice. (D, E) N wild-type = 6 mice, N SCA1-KI = 6 mice.

Article Snippet: To stain for small conductance calcium-activated potassium channel 2 (SK2) in IO ion channel studies , Rat anti-SK2 primary Ab (1:200, NeuroMab clone K78/29, Cat# 75-403, RRID:AB_2877597) and Goat anti-Rat IgG (H+L) Alexa Fluor 488-conjugated secondary Ab (1:200, Invitrogen, Cat# A11006, RRID:AB_2534074) were used.

Techniques: Expressing, Quantitative Proteomics, Immunostaining, Staining, Membrane, Derivative Assay

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: Details of primary and secondary antibodies.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques:

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: Immunoblot analyses of lysates from HEK293 and HeLa cells treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and lysates from wildtype (WT) or Sphk2 -/- MEFs. An equal amount (40 µg) of total protein from each sample was run in duplicate. After transferring to nitrocellulose and blocking, the membrane was separated and duplicate samples were probed with either ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. SK2 membranes were imaged using a 4 min exposure. The expected band size for SK2 is ∼65 kDa. Membranes were re-probed with mouse anti-α-tubulin antibody as a loading control (2 min exposure), which was detected at 55 kDa as expected. Consistent results were observed from 2-3 (HEK293 and MEF) or 3-4 (HeLa) independent experiments for each antibody.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Western Blot, Control, Transferring, Blocking Assay, Membrane

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: SK2 was immunoprecipitated from HEK293 cell lysate using either ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. Normal rabbit IgG antibody was used as an isotype control. Immunoprecipitates (and 40 µg lysate input) were subjected to immunoblot analyses and probed with ( A ) Proteintech rabbit anti-SK2 antibody or ( B ) ECM Biosciences rabbit anti-SK2 antibody. Membranes were imaged using a 4 min exposure. Images are representative of three independent experiments for each antibody. ( C ) SK2 was immunoprecipitated from HEK293 cell lysates (of equal protein) that had been treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), using ECM Biosciences rabbit anti-SK2 antibody. Immunoprecipitates were subjected to immunoblot analyses and probed with ECM Biosciences rabbit anti-SK2 antibody. Membrane was imaged using a 4 min exposure. Image is representative of three independent experiments. IgG h/c = IgG heavy chain.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Immunoprecipitation, Control, Western Blot, Membrane

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: ( A ) HeLa or ( B ) HEK293 cells were treated with scrambled control siRNA (si-Neg) or SK2 siRNA (si-SK2), and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. ( C ) Wildtype (WT) or Sphk2 -/- MEFs were seeded, and endogenous SK2 (green) was visualised by immunofluorescence staining and confocal microscopy, using Proteintech rabbit anti-SK2 antibody or ECM Biosciences rabbit anti-SK2 antibody. Nuclei were stained with DAPI (blue). For each cell line, background staining was examined by staining cells (si-Neg or WT cells) with secondary antibody and DAPI only, and collecting images using both 488nm and 405nm lasers (SK2 + DAPI). Images were taken at 40× magnification; scale bars = 10 µm. Images shown are representative of more than 100 cells from each experiment, and these results were consistent over three independent experiments for each cell line.

Article Snippet: Blots were probed with Proteintech rabbit polyclonal anti-SK2 antibody ( A–D ) or ECM Biosciences rabbit polyclonal anti-SK2 antibody ( E–H ).

Techniques: Control, Immunofluorescence, Staining, Confocal Microscopy

Journal: Science signaling

Article Title: A role for corticotrophin releasing factor signaling in the lateral habenula and its modulation by early life stress *

doi: 10.1126/scisignal.aan6480

Figure Lengend Snippet: MD increased the excitability of LHb neurons. A. Top: Representative spontaneous AP recordings of LHb neurons from non-MD and MD rats using cell-attached voltage clamp recordings. Bottom: Percent of spontaneously active LHb neurons (left) and mean AP frequency (right) from each group (n= 33 cells from 18 rats or 29 cells from 19 rats, respectively). B. Representative traces (top) and number of APs (bottom) recorded from LHb neurons using whole cell patch clamp recording, with synaptic transmission intact, in slices from non-MD and MD rats in response to depolarizing current steps (calibration bar: 20mV/1s) [n= 24 cells from 11 rats (non-MD) or 24 cells from 9 rats (MD), F(1,460)=1.69]C. As described in (B), with fast synaptic transmission blocked [n= 25 cells from 17 rats (non-MD) or 31 cells from 18 rats (MD), F(1,600)=35.84]. D: Average amplitude of fAHP, mAHP and Rin derived from AP recordings in (C). E: Representative Western blots and quantitation of SK2 abundance (β-actin: loading control) in LHb tissue homogenates from non-MD and MD rats (n= 6 biological replicates). Data are means ± SEM. *P<0.05, **P<0.01 by unpaired Student's t-test; ****P<0.0001 by two-way ANOVA.

Article Snippet: Separated proteins were transferred onto nitrocellulose membranes, blocked with casein-based blocking reagent (I-Block, Life Technologies) for 60 minutes at room temperature and then incubated overnight at 4°C with rabbit antibodies recognizing SKs (Antibody against KCa2.1 for SK1, Antibody against KCa2.2 for SK2, and Antibody against KCa2.3 (C-term) for SK3,1:300, Alomone Labs), antibody against PKA regulatory β2 subunit (1:5000, Abcam ab75993), antibody against BK slo-1 (α1 subunit of BK) (1:1000, Millipore MADN70) and GAPDH (1:1000, Cell signaling 2118) or antibody against actin (1:10,000, Abcam, ab6276).

Techniques: Patch Clamp, Transmission Assay, Derivative Assay, Western Blot, Quantitation Assay

Journal: F1000Research

Article Title: Validation of commercially available sphingosine kinase 2 antibodies for use in immunoblotting, immunoprecipitation and immunofluorescence

doi: 10.12688/f1000research.10336.2

Figure Lengend Snippet: Details of primary and secondary antibodies.

Article Snippet: The Proteintech anti-SK2 antibody produced minimal staining in all cell lines tested , and consequently there was no observable differences between the control cells and those with SK2 knockdown (in the human cell lines) or SK2 knockout (in the Sphk2 -/- MEF line).

Techniques: