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  • 99
    Millipore 2 apb
    Overview of pores in apo, sensitized and TRPV3 in the presence of <t>2-APB.</t> a Comparison of S6 helices of subunits A and C (top panel) and B and D (bottom panel) in apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres. b Bottom-up view of the pores of apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres
    2 Apb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Tocris 2 apb
    Effects of <t>2-APB,</t> a well-known SOCC blocker, on ACh-induced Ca 2+ oscillations. (A) A representative typical trace showing that application of 30 μmol/L congo red enhanced ACh (30 nmol/L)-induced Ca 2+ oscillations. (B) A representative typical
    2 Apb, supplied by Tocris, used in various techniques. Bioz Stars score: 97/100, based on 348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 2apb
    Orai3 silencing blocks <t>2APB</t> dependent increase in SOCE Calcium imaging was performed in control ( A ) and Orai3 knockdown ( B ) NMuMG cells. Analog plots of the fluorescence ratio (340/380) from an average of 40–60 cells are shown. ( C ) Quantification (mean ± SD) of fluorescence ratio (340/380). All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.
    2apb, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical 2 apb
    IP 3 -mediated calcium signaling participates in fractalkine-induced [Ca 2+ ] i release in BV-2 cells. ( A ) [Ca 2+ ] i was increased by fractalkine in the media with calcium. ( B ) [Ca 2+ ] i was increased by fractalkine in the media with calcium-free. ( C ) The elevation of [Ca 2+ ] i by fractalkine was inhibited by <t>2-APB.</t> * P
    2 Apb, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega 2 apb
    The effects of incorporation of ΔpCt or ΔCt on the gating processes induced by <t>2-APB.</t> (A) Diagram of a single TREK-2 subunit, transmembrane segments 1~4 (M1~M4), N-terminus, the proximal C-terminus (pCt), pore domain 1 (P1) and 2 (P2), glycine hinge of M2 (G196) and M4 (G312) are indicated. (B) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (C,D) Comparative analysis of the 2-APB evoked activation curves for the indicated channels. Due to the lower sensitivity of ΔpCt-ΔpCt to 2-APB, higher concentrations were used in its curve.
    2 Apb, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    2 apb  (Abcam)
    97
    Abcam 2 apb
    The effects of incorporation of ΔpCt or ΔCt on the gating processes induced by <t>2-APB.</t> (A) Diagram of a single TREK-2 subunit, transmembrane segments 1~4 (M1~M4), N-terminus, the proximal C-terminus (pCt), pore domain 1 (P1) and 2 (P2), glycine hinge of M2 (G196) and M4 (G312) are indicated. (B) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (C,D) Comparative analysis of the 2-APB evoked activation curves for the indicated channels. Due to the lower sensitivity of ΔpCt-ΔpCt to 2-APB, higher concentrations were used in its curve.
    2 Apb, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Robust LTD 2 apb
    Effect of <t>2-APB</t> on LTP induced by two successive short tetani. Summarized time-course data for LTP in S-EPSP ( A ) or A-PS ( B ) induced by two successive tetani of 10 pulses (T 10 ) and 15 pulses (T 15 ), given in the absence (Control, empty circles) or the
    2 Apb, supplied by Robust LTD, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    FUJIFILM 2 apb
    Abolition of fasiglifam-induced increase in nonselective cation channel (NSCC) current by inhibitors of the phospholipase (PLC)/protein kinase C (PKC) and TRP channels. ( a–c ) Effects of the nonselective transient receptor potential (TRP) channel blocker 2-aminoethyl diphenylborinate <t>(2-APB;</t> 10 μM) ( a ), TRP canonical (TRPC) channel blocker 3,5-bis(trifluoromethyl)pyrazole derivative 2 (BTP2; 10 μM) ( b ) and selective TRPC3 channel blocker pyrazole-3 (Pyr3; 10 μM) ( c ) on the NSCC current. ( d–f ) Effects of the PLC inhibitor U73122 (2 μM) ( d ) and PKC inhibitors Gö6983 (1 μM) ( e ) and Gö6976 (1 μM) ( f ) on the NSCC current. Fasiglifam-induced current increases were inhibited by these inhibitors. Rat single beta cells were voltage-clamped at −70 mV in the presence or absence of 10 μM fasiglifam, under the condition of 5.6 mM glucose and 100 μM tolbutamide throughout the experiments. The number of data points was five.
    2 Apb, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA 2 apb
    Effect of Ca 2+ channel inhibitors on calpain activity and T cell migration. Effect of ion channel inhibitors <t>2-APB</t> (50 µM), SKF-96365 (100 µM), LaCl 3 (2 mM) on the following ( A–D ): ( A ) Calpain activity as detected by expression of calpain substrate CMAC, t -BOC-Leu-Met. Pseudo-color glow scale (red, highest to blue, lowest activity). Outline of DIC image shown as a white tracing. Scale bar = 10 µm. Images are representative of n = 4 experiments; ( B ) Individual T cells show random migration following observation for 20 min (n = 15 cells, n = 3 experiments); ( C ) Overall speed of T cell migration (n = 15 cells). *** P
    2 Apb, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher 2 apb
    Knockdown of STIM1 attenuates SOCE in human NPCs. (A,B) Ca 2+ -responses during ER-store release and SOCE induced by Thapsigargin (TG, 10 μM) measured using the ratiometric Ca 2+ -indicator indo-1-AM in wild-type (WT) hNPCs (A) or hNPCs treated with pharmacological inhibitors of SOCE, BTP-2 and <t>2-APB</t> at the indicated concentrations or DMSO as a solvent control (B) . Each trace represents the mean ±SEM for 25–100 cells. Ionomycin (Iono, 10 μM) was added at the end of each imaging to determine the peak F405/485 ratio obtained after saturation of the Ca 2+ -indicator with Ca 2+ . (C) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated treatment conditions. Mann–Whitney U test with Bonferroni correction. p = 1.819 × 10 -23 for DMSO control compared to BTP-2 treatment and p = 1.442 × 10 -45 for DMSO control compared to 2-APB treatment. (D) (Top) A representative Western blot showing levels of STIM1 protein in hNPCs transduced with an NTC (non-targeting control) or an sh-RNA targeting STIM1 ( STIM1 KD). Actin serves as the loading control. (Bottom) Quantification of STIM1 band intensities normalized to the loading control Actin from three independent biological replicates ( p = 0.00069, Student’s t -test). (E) Ca 2+ -responses during store-release and SOCE in hNPCs transduced with NTC and STIM1 KD. (F) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated genotypes. Peak F405/485 for store-release were not significantly different between NTC and STIM1 KD NPCs. p = 0.0001 for peak F405/485 during SOCE compared between NTC- and STIM1 KD NPCs. (G) Quantification of basal cytosolic [Ca 2+ ] values using Fura-2-AM in NTC- and STIM1 KD NPCs ( p = 1.115 × 10 -8 . Mann–Whitney U test. ( ∗∗∗ Indicates p
    2 Apb, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of pores in apo, sensitized and TRPV3 in the presence of 2-APB. a Comparison of S6 helices of subunits A and C (top panel) and B and D (bottom panel) in apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres. b Bottom-up view of the pores of apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres

    Journal: Nature Communications

    Article Title: Conformational ensemble of the human TRPV3 ion channel

    doi: 10.1038/s41467-018-07117-w

    Figure Lengend Snippet: Overview of pores in apo, sensitized and TRPV3 in the presence of 2-APB. a Comparison of S6 helices of subunits A and C (top panel) and B and D (bottom panel) in apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres. b Bottom-up view of the pores of apo (gold), sensitized (cyan), TRPV3 2-APB 1–3 (magenta, red, and slate). Residues representing the helix bundle gate are shown as sticks and spheres

    Article Snippet: Glass coverslips with adherent transfected cells were placed into an open bath chamber (RC-26G, Warner Instruments) and an extracellular solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2 , 10 HEPES at pH 7.4 (NaOH) with and without (wash) 30 µM 2-APB (Sigma) (prepared daily from dimethyl sulfoxide (DMSO) stocks (1 M) stored at −80 °C; final DMSO 0.03%) was focally applied with a pressurized perfusion system (BPS-8, ALA Scientific Instruments).

    Techniques:

    Reduced symmetry in the structures of TRPV3 obtained in the presence of 2-APB a Overlay of sensitized TRPV3 (cyan) and TRPV3 2-APB 2 (red). Subunits B and D of TRPV3 2-APB 2 undergo a ~3° rotation. b Superposition of subunit B of sensitized TRPV3 (cyan) and TRPV3 2-APB 2 (red). The S4−S5 linker deviates from the alignment. c Overlay of subunits A (light blue) and B (red) of TRPV3 2-APB 2. The S4−S5 linkers in the two subunits assume different conformations. d Overlay of S5 helices of subunits A (light blue) and B (red) of TRPV3 2-APB 2. The two S4−S5 linkers have π-helices in different positions

    Journal: Nature Communications

    Article Title: Conformational ensemble of the human TRPV3 ion channel

    doi: 10.1038/s41467-018-07117-w

    Figure Lengend Snippet: Reduced symmetry in the structures of TRPV3 obtained in the presence of 2-APB a Overlay of sensitized TRPV3 (cyan) and TRPV3 2-APB 2 (red). Subunits B and D of TRPV3 2-APB 2 undergo a ~3° rotation. b Superposition of subunit B of sensitized TRPV3 (cyan) and TRPV3 2-APB 2 (red). The S4−S5 linker deviates from the alignment. c Overlay of subunits A (light blue) and B (red) of TRPV3 2-APB 2. The S4−S5 linkers in the two subunits assume different conformations. d Overlay of S5 helices of subunits A (light blue) and B (red) of TRPV3 2-APB 2. The two S4−S5 linkers have π-helices in different positions

    Article Snippet: Glass coverslips with adherent transfected cells were placed into an open bath chamber (RC-26G, Warner Instruments) and an extracellular solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2 , 10 HEPES at pH 7.4 (NaOH) with and without (wash) 30 µM 2-APB (Sigma) (prepared daily from dimethyl sulfoxide (DMSO) stocks (1 M) stored at −80 °C; final DMSO 0.03%) was focally applied with a pressurized perfusion system (BPS-8, ALA Scientific Instruments).

    Techniques:

    Structural and functional characterization of the human TRPV3. a 3D cryo-EM reconstruction of the apo human TRPV3 tetramer, colored by protomer. The C-terminal domain is shown in red. b Overview of the structural elements within a single protomer of the TRPV3 channel: Ankyrin repeat domain (ARD) is shown in yellow; Coupling domain (CD), consisting of linker domain, pre-S1 and the C-terminal domain, is shown in red; Voltage sensing-like domain (VSLD), consisting of helices S1−S4, is shown in blue, the pore domain, consisting of helices S5−S6 and the pore helix, is colored in purple; the TRP domain is shown in green. c , d Functional characterization of the TRPV3 wild-type ( c ) and T96A mutant ( d ) channels. Representative whole-cell current traces recorded at +60 mV from TRPV3 wild-type and T96A channels evoked by repeated application of 30 µM 2-APB for 15 s followed by complete washout (left) and corresponding time course of sensitization from the depicted recording (right) (TRPV3WT: n = 7 biologically independent experiments; TRPV3 T96A: n = 6 biologically independent experiments). e , f The half-maximal stimulation number ( S 50 ) ( e ) and rate of change ( k ) ( f ) of sensitization were calculated as the mean values obtained from fits of time course of sensitization plots from individual recordings (TRPV3WT: n = 7 biologically independent experiments; TRPV3 T96A: n = 6 biologically independent experiments) with standard two-state Boltzmann equation (see Methods). The extent of sensitization ( g ) was characterized by the relative increase in the current response to 2-APB during the first ( I 0 ) and maximum current ( I max ) stimulation ( I max / I 0 ) and calculated as the mean from each biologically independent experiment. No significant differences (NS) in the S 50 ( P = 0.86), k ( P = 0.46), and I max / I 0 ( P = 0.90) was determined between TRPV3WT and TRPV3 T96A (two-tailed Student’s t test, P > 0.05). Confidence intervals (95%): −14.2 (low)/12.2 (high) for S 50 , −2.54 (low)/1.52(high) for k , −55.1 (low)/49.8 (high) for I max / I 0 . Bar graphs and error bars denote mean ± s.e.m. The source data underlying Fig. 1e–g are provided as a Source Data file

    Journal: Nature Communications

    Article Title: Conformational ensemble of the human TRPV3 ion channel

    doi: 10.1038/s41467-018-07117-w

    Figure Lengend Snippet: Structural and functional characterization of the human TRPV3. a 3D cryo-EM reconstruction of the apo human TRPV3 tetramer, colored by protomer. The C-terminal domain is shown in red. b Overview of the structural elements within a single protomer of the TRPV3 channel: Ankyrin repeat domain (ARD) is shown in yellow; Coupling domain (CD), consisting of linker domain, pre-S1 and the C-terminal domain, is shown in red; Voltage sensing-like domain (VSLD), consisting of helices S1−S4, is shown in blue, the pore domain, consisting of helices S5−S6 and the pore helix, is colored in purple; the TRP domain is shown in green. c , d Functional characterization of the TRPV3 wild-type ( c ) and T96A mutant ( d ) channels. Representative whole-cell current traces recorded at +60 mV from TRPV3 wild-type and T96A channels evoked by repeated application of 30 µM 2-APB for 15 s followed by complete washout (left) and corresponding time course of sensitization from the depicted recording (right) (TRPV3WT: n = 7 biologically independent experiments; TRPV3 T96A: n = 6 biologically independent experiments). e , f The half-maximal stimulation number ( S 50 ) ( e ) and rate of change ( k ) ( f ) of sensitization were calculated as the mean values obtained from fits of time course of sensitization plots from individual recordings (TRPV3WT: n = 7 biologically independent experiments; TRPV3 T96A: n = 6 biologically independent experiments) with standard two-state Boltzmann equation (see Methods). The extent of sensitization ( g ) was characterized by the relative increase in the current response to 2-APB during the first ( I 0 ) and maximum current ( I max ) stimulation ( I max / I 0 ) and calculated as the mean from each biologically independent experiment. No significant differences (NS) in the S 50 ( P = 0.86), k ( P = 0.46), and I max / I 0 ( P = 0.90) was determined between TRPV3WT and TRPV3 T96A (two-tailed Student’s t test, P > 0.05). Confidence intervals (95%): −14.2 (low)/12.2 (high) for S 50 , −2.54 (low)/1.52(high) for k , −55.1 (low)/49.8 (high) for I max / I 0 . Bar graphs and error bars denote mean ± s.e.m. The source data underlying Fig. 1e–g are provided as a Source Data file

    Article Snippet: Glass coverslips with adherent transfected cells were placed into an open bath chamber (RC-26G, Warner Instruments) and an extracellular solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2 , 10 HEPES at pH 7.4 (NaOH) with and without (wash) 30 µM 2-APB (Sigma) (prepared daily from dimethyl sulfoxide (DMSO) stocks (1 M) stored at −80 °C; final DMSO 0.03%) was focally applied with a pressurized perfusion system (BPS-8, ALA Scientific Instruments).

    Techniques: Functional Assay, Mutagenesis, Two Tailed Test

    Effects of 2-APB, a well-known SOCC blocker, on ACh-induced Ca 2+ oscillations. (A) A representative typical trace showing that application of 30 μmol/L congo red enhanced ACh (30 nmol/L)-induced Ca 2+ oscillations. (B) A representative typical

    Journal: Acta Pharmacologica Sinica

    Article Title: Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

    doi: 10.1038/aps.2014.94

    Figure Lengend Snippet: Effects of 2-APB, a well-known SOCC blocker, on ACh-induced Ca 2+ oscillations. (A) A representative typical trace showing that application of 30 μmol/L congo red enhanced ACh (30 nmol/L)-induced Ca 2+ oscillations. (B) A representative typical

    Article Snippet: 2-APB was purchased from Tocris Bioscience (Minneapolis, MN USA).

    Techniques:

    The Zn 2+ -induced [Ca 2+ ] i increase is PLC-β-dependent. Fura-2-loaded HSG cells were treated with 30 µM ZnCl 2 ( a ), 300 µM carbachol ( b ), or 100 µM histamine ( c ) after pre-incubation with vehicle (left), 3 µM U73122 (middle), or 20 µM 2APB (left). Quantification of [Ca 2+ ] i increase inhibition mediated by carbachol or ZnCl 2 was normalized to vehicle groups. The experiments were performed three times independently, and the results were reproducible. Numbers of data were indicated inside bars. ** P

    Journal: Scientific Reports

    Article Title: Zn2+ stimulates salivary secretions via metabotropic zinc receptor ZnR/GPR39 in human salivary gland cells

    doi: 10.1038/s41598-019-54173-3

    Figure Lengend Snippet: The Zn 2+ -induced [Ca 2+ ] i increase is PLC-β-dependent. Fura-2-loaded HSG cells were treated with 30 µM ZnCl 2 ( a ), 300 µM carbachol ( b ), or 100 µM histamine ( c ) after pre-incubation with vehicle (left), 3 µM U73122 (middle), or 20 µM 2APB (left). Quantification of [Ca 2+ ] i increase inhibition mediated by carbachol or ZnCl 2 was normalized to vehicle groups. The experiments were performed three times independently, and the results were reproducible. Numbers of data were indicated inside bars. ** P

    Article Snippet: Pirenzepine, chlorpheniramine, U73122, and 2APB were obtained from Tocris (Bristol, UK).

    Techniques: Planar Chromatography, Incubation, Inhibition

    Orai3 silencing blocks 2APB dependent increase in SOCE Calcium imaging was performed in control ( A ) and Orai3 knockdown ( B ) NMuMG cells. Analog plots of the fluorescence ratio (340/380) from an average of 40–60 cells are shown. ( C ) Quantification (mean ± SD) of fluorescence ratio (340/380). All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.

    Journal: Oncotarget

    Article Title: The calcium channel proteins ORAI3 and STIM1 mediate TGF-β induced Snai1 expression

    doi: 10.18632/oncotarget.25672

    Figure Lengend Snippet: Orai3 silencing blocks 2APB dependent increase in SOCE Calcium imaging was performed in control ( A ) and Orai3 knockdown ( B ) NMuMG cells. Analog plots of the fluorescence ratio (340/380) from an average of 40–60 cells are shown. ( C ) Quantification (mean ± SD) of fluorescence ratio (340/380). All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.

    Article Snippet: Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment.

    Techniques: Imaging, Fluorescence, Software

    Reversal of gene expression comparing TGF-β to TGF-β+2APB treatments ( A ) RNA isolated from NMuMG cells treated with DMSO, TGF-β or TGF-β+2APB was sequenced, and gene expression changes calculated (see Supplementary Figure 1 and methods for details). Using this gene list, we extracted genes with expression changes when treated with TGF-β that were reversed by the addition of 2APB. We selected genes that had a range of ratio values of –1.5 to –0.5 and hierarchical clustering of their Pearson correlation values was performed using the aheatmap function in the R package NMF v0.20.6. ( B ) Genes that were upregulated with TGF-β addition, and then downregulated with 2APB addition were analyzed using IPA. The top five scoring hits in these categories using IPA are shown, together with significance scores ( p -values) and the number of genes included in each class. A similar analysis was conducted with genes that were downregulated with TGF-β, but then reversed (up-regulated) with 2APB, and analyzed with IPA as above. ( C ) A subset of EMT-linked genes and their fold changes with TGF-β and TGF-β+2APB.

    Journal: Oncotarget

    Article Title: The calcium channel proteins ORAI3 and STIM1 mediate TGF-β induced Snai1 expression

    doi: 10.18632/oncotarget.25672

    Figure Lengend Snippet: Reversal of gene expression comparing TGF-β to TGF-β+2APB treatments ( A ) RNA isolated from NMuMG cells treated with DMSO, TGF-β or TGF-β+2APB was sequenced, and gene expression changes calculated (see Supplementary Figure 1 and methods for details). Using this gene list, we extracted genes with expression changes when treated with TGF-β that were reversed by the addition of 2APB. We selected genes that had a range of ratio values of –1.5 to –0.5 and hierarchical clustering of their Pearson correlation values was performed using the aheatmap function in the R package NMF v0.20.6. ( B ) Genes that were upregulated with TGF-β addition, and then downregulated with 2APB addition were analyzed using IPA. The top five scoring hits in these categories using IPA are shown, together with significance scores ( p -values) and the number of genes included in each class. A similar analysis was conducted with genes that were downregulated with TGF-β, but then reversed (up-regulated) with 2APB, and analyzed with IPA as above. ( C ) A subset of EMT-linked genes and their fold changes with TGF-β and TGF-β+2APB.

    Article Snippet: Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment.

    Techniques: Expressing, Isolation, Indirect Immunoperoxidase Assay

    2APB increases TGF-β dependent activation of the AKT pathway and recruitment of NF-κB and Pol II to the Snai1 promoter ( A ) NMuMG cells were serum starved for 4 hours and then treated with 2APB for 24 hours. At 18 hours, the cells were treated with 50 uM of the NFKB inhibitor ACHP for a 4-hour pretreatment before addition of TGF-β at 22 hours. ( B ) NMuMG cells were serum starved for 4 h and then treated with 10 uM of the inhibitor at 20 hours for a 2-hour pretreatment before the TGF-β treatment at 22 hours for 2 hours. ( C ) NMuMG cells were serum-starved for 4 h, and then treated with DMSO, 2APB, TGF-β or TGF-β+2APB for 24 h. Protein isolation from these cells followed by immunoblotting using antibodies to phospho- AKT Ser473 , total AKT, and GAPDH. Data are representative of 3–4 independent biological replicates. Bar graphs next to the image represent the quantitation of blots using LiCOR image software, and statistical analyses performed using GraphPad Prism. * = p -value ≤ 0.05. ( D ) The samples from (C) were immunoblotted for phospho-p65 Ser536 , total p65 (RelA subunit of NF-κΒ), and normalized to GAPDH as above. ( E ) Schematic representation of the primer sets used in chromatin IP covering ~3.8 Kb of the Snai1 promoter region. The putative NF-κΒ binding sites are depicted as vertical lines. Locations of primer sets are indicated below the gene, as are the distances between the primer pairs in bp. ( F ) Chromatin immunoprecipitation (ChIP) was performed using antibodies against Pol II and p65, with IgG as a negative control. real-time PCR amplification of ChIP DNA across the Snai1 locus reveals a peak of Pol II over the promoter region encompassed by primer set ‘Prom.’ in cells treated with TGF-β (white bars). This peak increases with 2APB treatment (grey bars). No discernable Pol II signal is noted in the vehicle (DMSO) treated cells (black bars). ( G ) While no p65 is apparent in DMSO treated cells, there is increased association of p65 at the Snai1 promoter DNA with addition of TGF-β and with TGF-β+2APB. All data are representative of three independent biological replicates.

    Journal: Oncotarget

    Article Title: The calcium channel proteins ORAI3 and STIM1 mediate TGF-β induced Snai1 expression

    doi: 10.18632/oncotarget.25672

    Figure Lengend Snippet: 2APB increases TGF-β dependent activation of the AKT pathway and recruitment of NF-κB and Pol II to the Snai1 promoter ( A ) NMuMG cells were serum starved for 4 hours and then treated with 2APB for 24 hours. At 18 hours, the cells were treated with 50 uM of the NFKB inhibitor ACHP for a 4-hour pretreatment before addition of TGF-β at 22 hours. ( B ) NMuMG cells were serum starved for 4 h and then treated with 10 uM of the inhibitor at 20 hours for a 2-hour pretreatment before the TGF-β treatment at 22 hours for 2 hours. ( C ) NMuMG cells were serum-starved for 4 h, and then treated with DMSO, 2APB, TGF-β or TGF-β+2APB for 24 h. Protein isolation from these cells followed by immunoblotting using antibodies to phospho- AKT Ser473 , total AKT, and GAPDH. Data are representative of 3–4 independent biological replicates. Bar graphs next to the image represent the quantitation of blots using LiCOR image software, and statistical analyses performed using GraphPad Prism. * = p -value ≤ 0.05. ( D ) The samples from (C) were immunoblotted for phospho-p65 Ser536 , total p65 (RelA subunit of NF-κΒ), and normalized to GAPDH as above. ( E ) Schematic representation of the primer sets used in chromatin IP covering ~3.8 Kb of the Snai1 promoter region. The putative NF-κΒ binding sites are depicted as vertical lines. Locations of primer sets are indicated below the gene, as are the distances between the primer pairs in bp. ( F ) Chromatin immunoprecipitation (ChIP) was performed using antibodies against Pol II and p65, with IgG as a negative control. real-time PCR amplification of ChIP DNA across the Snai1 locus reveals a peak of Pol II over the promoter region encompassed by primer set ‘Prom.’ in cells treated with TGF-β (white bars). This peak increases with 2APB treatment (grey bars). No discernable Pol II signal is noted in the vehicle (DMSO) treated cells (black bars). ( G ) While no p65 is apparent in DMSO treated cells, there is increased association of p65 at the Snai1 promoter DNA with addition of TGF-β and with TGF-β+2APB. All data are representative of three independent biological replicates.

    Article Snippet: Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment.

    Techniques: Activation Assay, Isolation, Quantitation Assay, Software, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Amplification

    Orai3 silencing inhibits both cell migration and Snai1 transcription in response to TGF-β ( A ) NMuMG cells were treated as indicated with TGF-β, TGF-β+2APB or DMSO, in the presence or absence of siOrai3 and RNA isolated. RNA was converted to cDNA and analyzed by real-time PCR using primers specific to mouse Snai1, Snai2 or Twist1, and normalized to Gapdh. Data represent the average of 3 individual biological replicates. ( B ) Proteins isolated from the same cells as in (A) were evaluated for SNAI1 expression by immunoblotting. Antibody to ACTIN was used a loading control, and the blots are representative of at least 3 independent biological replicates. Blots were quantitated using the LiCOR imaging software and are represented as SNAI1/ACTB signal, after normalizing to DMSO control. Error bars represent SEM and statistical analyses were performed Graphpad PRISM. * = p -value ≤ 0.05, ** = p -value ≤ 0.01 relative to control. ( C ) Confluent NMuMG cells in a 6-well plate were serum starved for 4 h prior to treatment, and TGF-β (for 8 h) and/or 2APB (for 24 h) were added to the wells prior to wounding using a sterile 200 ul tip. Three representative fields were marked and imaged immediately at time of (0 h) and a time period after (8 h) wounding as described in materials and methods. The images were captured using an Olympus IX71 microscope camera. All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05, ** = p -value ≤ 0.01; *** = p -value ≤ 0.001; **** = p -value ≤ 0.0001. ( D ) Model for ORAI3-mediated Snai1 upregulation. AKT (green oval) pathway can be activated by both calcium (black circles) and by TGF-β signaling. 2APB prevents SOCE via ORAI1 and ORAI2, while increasing calcium influx through ORAI3. Activation of AKT triggers increased binding of p65 at the Snai1 promoter, leading to increased recruitment of Pol II and hence transcription of Snai1 .

    Journal: Oncotarget

    Article Title: The calcium channel proteins ORAI3 and STIM1 mediate TGF-β induced Snai1 expression

    doi: 10.18632/oncotarget.25672

    Figure Lengend Snippet: Orai3 silencing inhibits both cell migration and Snai1 transcription in response to TGF-β ( A ) NMuMG cells were treated as indicated with TGF-β, TGF-β+2APB or DMSO, in the presence or absence of siOrai3 and RNA isolated. RNA was converted to cDNA and analyzed by real-time PCR using primers specific to mouse Snai1, Snai2 or Twist1, and normalized to Gapdh. Data represent the average of 3 individual biological replicates. ( B ) Proteins isolated from the same cells as in (A) were evaluated for SNAI1 expression by immunoblotting. Antibody to ACTIN was used a loading control, and the blots are representative of at least 3 independent biological replicates. Blots were quantitated using the LiCOR imaging software and are represented as SNAI1/ACTB signal, after normalizing to DMSO control. Error bars represent SEM and statistical analyses were performed Graphpad PRISM. * = p -value ≤ 0.05, ** = p -value ≤ 0.01 relative to control. ( C ) Confluent NMuMG cells in a 6-well plate were serum starved for 4 h prior to treatment, and TGF-β (for 8 h) and/or 2APB (for 24 h) were added to the wells prior to wounding using a sterile 200 ul tip. Three representative fields were marked and imaged immediately at time of (0 h) and a time period after (8 h) wounding as described in materials and methods. The images were captured using an Olympus IX71 microscope camera. All data are representative of at least 3 biological replicates. Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05, ** = p -value ≤ 0.01; *** = p -value ≤ 0.001; **** = p -value ≤ 0.0001. ( D ) Model for ORAI3-mediated Snai1 upregulation. AKT (green oval) pathway can be activated by both calcium (black circles) and by TGF-β signaling. 2APB prevents SOCE via ORAI1 and ORAI2, while increasing calcium influx through ORAI3. Activation of AKT triggers increased binding of p65 at the Snai1 promoter, leading to increased recruitment of Pol II and hence transcription of Snai1 .

    Article Snippet: Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment.

    Techniques: Migration, Isolation, Real-time Polymerase Chain Reaction, Expressing, Imaging, Software, Microscopy, Activation Assay, Binding Assay

    2APB amplifies the TGF-β dependent up-regulation of Snai1 transcription NMuMG cells were serum-starved for 4 h, and then treated with DMSO or 2APB for 24 h, and TGF-β for 2 ( A ), 8 ( B ) and 24 ( C ) hours. RNA was isolated from NMuMG cells and cDNA prepared using reverse transcription. Expression of EMT genes was examined by real-time PCR of the cDNA using primers against each of the genes and normalized to 18S rRNA . Data were derived from at least three independent biological replicates, and are represented as mean ± SEM values. The * indicates p -value of ≤ 0.05, and *** indicates p -value ≤ 0.001 as measured by a paired t -test. ( D ) Protein analysis of NMuMG lysates treated with DMSO, or 2APB for 24 h, and with TGF-β for 8 h (added after 16 h of treatment with 2APB) as above was performed using western blotting with antibodies against SNAI1 and ACTIN. The blot is representative of at least 3 independent biological replicates. Quantitation was performed as described in methods, normalizing the signal to ACTB loading control. ( E ) Expression of E-cadherin, a downstream target of all the above EMT was measured from the same time points as in (A, B and C). ( F ) To test whether the increase seen in Snai1 expression is due to increase in transcription, cells were treated with DMSO or 2APB for 24 h, followed by TGF-β, and Actinomycin D was added 2 h after addition of TGF-β for 1 h. RNA isolation was followed in a time course of up to 2 h after Actinomycin D treatment. RNA was converted to cDNA and Snai1 expression measured as in (A, B, C and E). Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.

    Journal: Oncotarget

    Article Title: The calcium channel proteins ORAI3 and STIM1 mediate TGF-β induced Snai1 expression

    doi: 10.18632/oncotarget.25672

    Figure Lengend Snippet: 2APB amplifies the TGF-β dependent up-regulation of Snai1 transcription NMuMG cells were serum-starved for 4 h, and then treated with DMSO or 2APB for 24 h, and TGF-β for 2 ( A ), 8 ( B ) and 24 ( C ) hours. RNA was isolated from NMuMG cells and cDNA prepared using reverse transcription. Expression of EMT genes was examined by real-time PCR of the cDNA using primers against each of the genes and normalized to 18S rRNA . Data were derived from at least three independent biological replicates, and are represented as mean ± SEM values. The * indicates p -value of ≤ 0.05, and *** indicates p -value ≤ 0.001 as measured by a paired t -test. ( D ) Protein analysis of NMuMG lysates treated with DMSO, or 2APB for 24 h, and with TGF-β for 8 h (added after 16 h of treatment with 2APB) as above was performed using western blotting with antibodies against SNAI1 and ACTIN. The blot is representative of at least 3 independent biological replicates. Quantitation was performed as described in methods, normalizing the signal to ACTB loading control. ( E ) Expression of E-cadherin, a downstream target of all the above EMT was measured from the same time points as in (A, B and C). ( F ) To test whether the increase seen in Snai1 expression is due to increase in transcription, cells were treated with DMSO or 2APB for 24 h, followed by TGF-β, and Actinomycin D was added 2 h after addition of TGF-β for 1 h. RNA isolation was followed in a time course of up to 2 h after Actinomycin D treatment. RNA was converted to cDNA and Snai1 expression measured as in (A, B, C and E). Statistical analyses were performed with Graphpad Prism software. * = p -value ≤ 0.05.

    Article Snippet: Cells were treated with 2APB (50 uM final; Sigma-Aldrich) for a period of 24 h prior to TGF-β treatment.

    Techniques: Isolation, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Western Blot, Quantitation Assay, Software

    Effect of retinoschisin on Ca 2+ signaling. (A) Rs1h -/Y retinal explants were treated for 10 and 30 min with retinoschisin, RS1-C59S, or control protein. After treatment, the cells were subjected to Western blot analyses with antibodies against phosphorylated Camk2 (pCamk2), total Camk2, and Actb as control. Densitometric quantification was performed with immunoblots from five independent experiments. Signals for pCamk2 and Camk2 were normalized against Actb and calibrated against the control. Data represent mean ± SD. (B) Rs1h -/Y retinal explants were treated for 30 min with Ca 2+ signaling (IP3 receptor) inhibitor 2-APB. To assess an effect of the Ca 2+ pathway on Erk1/2 activation, the cells were subjected to Western blot analyses with antibodies against phosphorylated Erk1 and Erk2 (pErk1/2), total Erk1 and Erk2 (Erk1/2), and Actb as control. Underlined asterisk marks statistically significant ( p

    Journal: Molecular Biology of the Cell

    Article Title: Retinoschisin is linked to retinal Na/K-ATPase signaling and localization

    doi: 10.1091/mbc.E17-01-0064

    Figure Lengend Snippet: Effect of retinoschisin on Ca 2+ signaling. (A) Rs1h -/Y retinal explants were treated for 10 and 30 min with retinoschisin, RS1-C59S, or control protein. After treatment, the cells were subjected to Western blot analyses with antibodies against phosphorylated Camk2 (pCamk2), total Camk2, and Actb as control. Densitometric quantification was performed with immunoblots from five independent experiments. Signals for pCamk2 and Camk2 were normalized against Actb and calibrated against the control. Data represent mean ± SD. (B) Rs1h -/Y retinal explants were treated for 30 min with Ca 2+ signaling (IP3 receptor) inhibitor 2-APB. To assess an effect of the Ca 2+ pathway on Erk1/2 activation, the cells were subjected to Western blot analyses with antibodies against phosphorylated Erk1 and Erk2 (pErk1/2), total Erk1 and Erk2 (Erk1/2), and Actb as control. Underlined asterisk marks statistically significant ( p

    Article Snippet: Src kinase inhibitor PP2 and IP3 receptor inhibitor 2-APB were obtained from Sigma- Aldrich and added at a final concentration of 10 or 250 µM, respectively.

    Techniques: Western Blot, Activation Assay

    IP 3 -mediated calcium signaling participates in fractalkine-induced [Ca 2+ ] i release in BV-2 cells. ( A ) [Ca 2+ ] i was increased by fractalkine in the media with calcium. ( B ) [Ca 2+ ] i was increased by fractalkine in the media with calcium-free. ( C ) The elevation of [Ca 2+ ] i by fractalkine was inhibited by 2-APB. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response

    doi: 10.12659/MSM.913787

    Figure Lengend Snippet: IP 3 -mediated calcium signaling participates in fractalkine-induced [Ca 2+ ] i release in BV-2 cells. ( A ) [Ca 2+ ] i was increased by fractalkine in the media with calcium. ( B ) [Ca 2+ ] i was increased by fractalkine in the media with calcium-free. ( C ) The elevation of [Ca 2+ ] i by fractalkine was inhibited by 2-APB. * P

    Article Snippet: In contrast, when treated with 2-APB prior to fractalkine addition, production of these factors was greatly decreased at 24 h, but 2-APB alone did not affect IL-1β or TNF-α protein levels ( ).

    Techniques:

    Fractalkine injection lead to thermal hyperalgesia and activated microglia in vivo . ( A ) The thermal nociceptive threshold of mice after receiving fractalkine, 2-APB, antiCX3CR1, and SB203580. ( B ) The immunofluorescence of FKN (fractalkine), Iba-1, and GFAP in mice brain tissues without or with fractalkine. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response

    doi: 10.12659/MSM.913787

    Figure Lengend Snippet: Fractalkine injection lead to thermal hyperalgesia and activated microglia in vivo . ( A ) The thermal nociceptive threshold of mice after receiving fractalkine, 2-APB, antiCX3CR1, and SB203580. ( B ) The immunofluorescence of FKN (fractalkine), Iba-1, and GFAP in mice brain tissues without or with fractalkine. * P

    Article Snippet: In contrast, when treated with 2-APB prior to fractalkine addition, production of these factors was greatly decreased at 24 h, but 2-APB alone did not affect IL-1β or TNF-α protein levels ( ).

    Techniques: Injection, In Vivo, Mouse Assay, Immunofluorescence

    Fractalkine upregulated pro-inflammatory cytokines in vivo. ( A, B ) The increase of IL-1β and TNF-α by treatment with fractalkine in RT-PCR analysis. ( C, D ) The increase of IL-1β and TNF-α by treatment with fractalkine in ELISA analysis. ( E, F ) The decrease of IL-1β and TNF-α by treatment with anti-CX3CR1, 2-APB, and SB203580. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response

    doi: 10.12659/MSM.913787

    Figure Lengend Snippet: Fractalkine upregulated pro-inflammatory cytokines in vivo. ( A, B ) The increase of IL-1β and TNF-α by treatment with fractalkine in RT-PCR analysis. ( C, D ) The increase of IL-1β and TNF-α by treatment with fractalkine in ELISA analysis. ( E, F ) The decrease of IL-1β and TNF-α by treatment with anti-CX3CR1, 2-APB, and SB203580. * P

    Article Snippet: In contrast, when treated with 2-APB prior to fractalkine addition, production of these factors was greatly decreased at 24 h, but 2-APB alone did not affect IL-1β or TNF-α protein levels ( ).

    Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Fractalkine upregulated p-p38MAPK protein in vivo. ( A ) The p-p38MAPK protein was increased after treatment with fractalkine. ( B ) The p-p38MAPK protein was attenuated by pretreatment with anti-CX3CR1, 2-APB, and SB203580. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response

    doi: 10.12659/MSM.913787

    Figure Lengend Snippet: Fractalkine upregulated p-p38MAPK protein in vivo. ( A ) The p-p38MAPK protein was increased after treatment with fractalkine. ( B ) The p-p38MAPK protein was attenuated by pretreatment with anti-CX3CR1, 2-APB, and SB203580. * P

    Article Snippet: In contrast, when treated with 2-APB prior to fractalkine addition, production of these factors was greatly decreased at 24 h, but 2-APB alone did not affect IL-1β or TNF-α protein levels ( ).

    Techniques: In Vivo

    IP 3 -mediated calcium signaling is involved in fractalkine-induced inflammatory responses in vitro. ( A, B ) The increased of IL-1β and TNF-α by exposed to fractalkine persistently. ( C, D ) The increased of IL-1β and TNF-α was decreased by 2-APB. ( E, F ) The mRNA of IL-1β and TNF-α were increased by exposed to fractalkine persistently. ( G, H ) The mRNA of IL-1β and TNF-α were decreased by 2-APB. * P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: IP3-Mediated Calcium Signaling Is Involved in the Mechanism of Fractalkine-Induced Hyperalgesia Response

    doi: 10.12659/MSM.913787

    Figure Lengend Snippet: IP 3 -mediated calcium signaling is involved in fractalkine-induced inflammatory responses in vitro. ( A, B ) The increased of IL-1β and TNF-α by exposed to fractalkine persistently. ( C, D ) The increased of IL-1β and TNF-α was decreased by 2-APB. ( E, F ) The mRNA of IL-1β and TNF-α were increased by exposed to fractalkine persistently. ( G, H ) The mRNA of IL-1β and TNF-α were decreased by 2-APB. * P

    Article Snippet: In contrast, when treated with 2-APB prior to fractalkine addition, production of these factors was greatly decreased at 24 h, but 2-APB alone did not affect IL-1β or TNF-α protein levels ( ).

    Techniques: In Vitro

    Effects of kinase and AA metabolic blockers on 2APB-elicited TRPV3 current in Xenopus oocytes. A : A representative trace showing that KN-62 did not affect the potentiation effect of AA in oocytes that expressed TRPV3. Cells were held at −40 mV

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Effects of kinase and AA metabolic blockers on 2APB-elicited TRPV3 current in Xenopus oocytes. A : A representative trace showing that KN-62 did not affect the potentiation effect of AA in oocytes that expressed TRPV3. Cells were held at −40 mV

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques:

    Effect of different free fatty acids on 2APB-evoked TRPV3 current in Xenopus oocytes. A : Oocytes expressing TRPV3 were held at −40 mV and stimulated with 300 μM 2APB without or with the pretreatment of 10 μM different free fatty

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Effect of different free fatty acids on 2APB-evoked TRPV3 current in Xenopus oocytes. A : Oocytes expressing TRPV3 were held at −40 mV and stimulated with 300 μM 2APB without or with the pretreatment of 10 μM different free fatty

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Expressing

    Potentiation of 2APB-evoked TRPV3 currents by AA in Xenopus oocytes. A : Consecutive responses of an oocyte that expressed TRPV3 to 3 min stimulations with 100 μM 2APB (open bars). AA (10 μM, black bar) was included for 1 min during the

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Potentiation of 2APB-evoked TRPV3 currents by AA in Xenopus oocytes. A : Consecutive responses of an oocyte that expressed TRPV3 to 3 min stimulations with 100 μM 2APB (open bars). AA (10 μM, black bar) was included for 1 min during the

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques:

    AA potentiates 2APB-evoked currents in HEK293 cells expressing TRPV3 but not those expressing TRPV1 or TRPV2. A : TRPV3-transfected HEK293 cells were stimulated with 2APB multiple times to establish a basal response level to 2APB. Then AA was applied continuously

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: AA potentiates 2APB-evoked currents in HEK293 cells expressing TRPV3 but not those expressing TRPV1 or TRPV2. A : TRPV3-transfected HEK293 cells were stimulated with 2APB multiple times to establish a basal response level to 2APB. Then AA was applied continuously

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Expressing, Transfection

    Properties of AA-potentiated TRPV3 currents. A–D , effects of 10 μM ruthenium red (RR) on the currents evoked by AA and 2APB in TRPV3-transfected cells. Shown are representative traces for membrane currents (A) at +100 (filled circles)

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Properties of AA-potentiated TRPV3 currents. A–D , effects of 10 μM ruthenium red (RR) on the currents evoked by AA and 2APB in TRPV3-transfected cells. Shown are representative traces for membrane currents (A) at +100 (filled circles)

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Transfection

    Potentiation by AA of single channel activity in patches excised from HEK293 cells expressing TRPV3. A : Activity in an inside-out patch held at −100 mV. 2APB and AA were added as indicated. Shown are NPo in 2-sec bins ( upper ), raw current trace

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Potentiation by AA of single channel activity in patches excised from HEK293 cells expressing TRPV3. A : Activity in an inside-out patch held at −100 mV. 2APB and AA were added as indicated. Shown are NPo in 2-sec bins ( upper ), raw current trace

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Activity Assay, Expressing, Size-exclusion Chromatography

    Concentration dependence of 2APB-evoked TRPV3 currents and its potentiation by AA in Xenopus oocytes. A : Representative traces showing the dose-dependent potentiation by AA of the response to 100 μM 2APB in an occyte that expressed TRPV3. B : Dose

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Concentration dependence of 2APB-evoked TRPV3 currents and its potentiation by AA in Xenopus oocytes. A : Representative traces showing the dose-dependent potentiation by AA of the response to 100 μM 2APB in an occyte that expressed TRPV3. B : Dose

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Concentration Assay

    Potentiation of 2APB-evoked TRPV3 currents by PMA in HEK293 cells. A : Similar experiment as in except the TRPV3-expressing cell was treated with 0.1 μM PMA for 40 sec as indicated. B : Similar to (A) except the cell was pretreated with

    Journal: Journal of cellular physiology

    Article Title: Potentiation of TRPV3 Channel Function by Unsaturated Fatty Acids

    doi: 10.1002/jcp.20648

    Figure Lengend Snippet: Potentiation of 2APB-evoked TRPV3 currents by PMA in HEK293 cells. A : Similar experiment as in except the TRPV3-expressing cell was treated with 0.1 μM PMA for 40 sec as indicated. B : Similar to (A) except the cell was pretreated with

    Article Snippet: Linoleic acid, docosahexaenoic acid, α-linolenic acid, γ-linolenic acid, 5,8,11,14-eicosate traynoic acid (ETYA), nordihydroguaiaretic acid (NDGA), and 2APB were purchased from Cayman Chemical Inc. (Ann Arbor, MI).

    Techniques: Expressing, Size-exclusion Chromatography

    The effects of incorporation of ΔpCt or ΔCt on the gating processes induced by 2-APB. (A) Diagram of a single TREK-2 subunit, transmembrane segments 1~4 (M1~M4), N-terminus, the proximal C-terminus (pCt), pore domain 1 (P1) and 2 (P2), glycine hinge of M2 (G196) and M4 (G312) are indicated. (B) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (C,D) Comparative analysis of the 2-APB evoked activation curves for the indicated channels. Due to the lower sensitivity of ΔpCt-ΔpCt to 2-APB, higher concentrations were used in its curve.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2

    doi: 10.3389/fncel.2016.00127

    Figure Lengend Snippet: The effects of incorporation of ΔpCt or ΔCt on the gating processes induced by 2-APB. (A) Diagram of a single TREK-2 subunit, transmembrane segments 1~4 (M1~M4), N-terminus, the proximal C-terminus (pCt), pore domain 1 (P1) and 2 (P2), glycine hinge of M2 (G196) and M4 (G312) are indicated. (B) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (C,D) Comparative analysis of the 2-APB evoked activation curves for the indicated channels. Due to the lower sensitivity of ΔpCt-ΔpCt to 2-APB, higher concentrations were used in its curve.

    Article Snippet: AR was calculated from I2-APB /Io , where I2-APB represented the currents in the presence of 2-APB, and Io was the baseline, stabilized currents in the absence of 2-APB, and was plotted as function of the concentration of 2-APB ([2-APB]o ).

    Techniques: Expressing, Activation Assay

    The effects of further introduction of G312A and ΔpCt, either individually or simultaneously, into the WT subunit of G312A-WT on the gating processes induced by 2-APB and extracellular alkalization. (A) Exemplar current-voltage recordings from oocytes expressing G312A-ΔpCt and G312A-G312A/ΔpCt channels in the presence of 100 μM 2-APB. (B) Concentration responses of indicated channels activated by 2-APB. *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2

    doi: 10.3389/fncel.2016.00127

    Figure Lengend Snippet: The effects of further introduction of G312A and ΔpCt, either individually or simultaneously, into the WT subunit of G312A-WT on the gating processes induced by 2-APB and extracellular alkalization. (A) Exemplar current-voltage recordings from oocytes expressing G312A-ΔpCt and G312A-G312A/ΔpCt channels in the presence of 100 μM 2-APB. (B) Concentration responses of indicated channels activated by 2-APB. *** p

    Article Snippet: AR was calculated from I2-APB /Io , where I2-APB represented the currents in the presence of 2-APB, and Io was the baseline, stabilized currents in the absence of 2-APB, and was plotted as function of the concentration of 2-APB ([2-APB]o ).

    Techniques: Expressing, Concentration Assay

    Concatenated wild type (WT) TREK-2 dimers (WT-WT) functions similarly with their monomeric WT channels (TREK-2). (A) Diagram of concatenated TREK-2 dimer and the sequence of linker indicated. (B) Validation of TREK-2 and WT-WT proteins was tested by western blotting. cDNA encoding TREK-2 or WT-WT was injected into oocytes. The monomeric TREK-2 was indicated with red arrow, and the dimeric one with green arrow. *Indicates the nonspecific bands detected by the anti-TREK-2 primary antibody in oocytes (left panel). Constructs harboring GFP-tagged TREK-2 or WT-WT was transfected into HEK293 cells (right panel). (C) Exemplar current-voltage recordings from oocytes expressing the WT-WT channels in the presence of 100 μM 2-APB. The currents were elicited by continuous voltage-ramps from −120 to +60 mV from holding potential of −80 mV, with 2 s in duration. (D) Comparative analysis of the 2-APB evoked activation curves between TREK-2 and WT-WT channels. (E) Exemplar current-voltage recordings from oocytes expressing WT-WT channels as pH o transitions among 6.5, 8.0 and 9.3. (F) Concentration dependence of Inhibition ratio (IR) upon extracellular alkalization for TREK-2 and WT-WT channels.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2

    doi: 10.3389/fncel.2016.00127

    Figure Lengend Snippet: Concatenated wild type (WT) TREK-2 dimers (WT-WT) functions similarly with their monomeric WT channels (TREK-2). (A) Diagram of concatenated TREK-2 dimer and the sequence of linker indicated. (B) Validation of TREK-2 and WT-WT proteins was tested by western blotting. cDNA encoding TREK-2 or WT-WT was injected into oocytes. The monomeric TREK-2 was indicated with red arrow, and the dimeric one with green arrow. *Indicates the nonspecific bands detected by the anti-TREK-2 primary antibody in oocytes (left panel). Constructs harboring GFP-tagged TREK-2 or WT-WT was transfected into HEK293 cells (right panel). (C) Exemplar current-voltage recordings from oocytes expressing the WT-WT channels in the presence of 100 μM 2-APB. The currents were elicited by continuous voltage-ramps from −120 to +60 mV from holding potential of −80 mV, with 2 s in duration. (D) Comparative analysis of the 2-APB evoked activation curves between TREK-2 and WT-WT channels. (E) Exemplar current-voltage recordings from oocytes expressing WT-WT channels as pH o transitions among 6.5, 8.0 and 9.3. (F) Concentration dependence of Inhibition ratio (IR) upon extracellular alkalization for TREK-2 and WT-WT channels.

    Article Snippet: AR was calculated from I2-APB /Io , where I2-APB represented the currents in the presence of 2-APB, and Io was the baseline, stabilized currents in the absence of 2-APB, and was plotted as function of the concentration of 2-APB ([2-APB]o ).

    Techniques: Sequencing, Western Blot, Injection, Construct, Transfection, Expressing, Activation Assay, Concentration Assay, Inhibition

    The effects of incorporation of G312A on the gating processes induced by 2-APB and extracellular alkalization. (A) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (B) Concentration responses of indicated channels activated by 2-APB. (C) Exemplar current-voltage recordings from oocytes expressing the indicated channels as pH o transitions among 6.5, 8.0 and 9.3. (D) Comparative analysis of pH o -inhibition curves for the indicated channels.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Intersubunit Concerted Cooperative and cis-Type Mechanisms Modulate Allosteric Gating in Two-Pore-Domain Potassium Channel TREK-2

    doi: 10.3389/fncel.2016.00127

    Figure Lengend Snippet: The effects of incorporation of G312A on the gating processes induced by 2-APB and extracellular alkalization. (A) Exemplar current-voltage recordings from oocytes expressing the indicated channels in the presence of 100 μM 2-APB. (B) Concentration responses of indicated channels activated by 2-APB. (C) Exemplar current-voltage recordings from oocytes expressing the indicated channels as pH o transitions among 6.5, 8.0 and 9.3. (D) Comparative analysis of pH o -inhibition curves for the indicated channels.

    Article Snippet: AR was calculated from I2-APB /Io , where I2-APB represented the currents in the presence of 2-APB, and Io was the baseline, stabilized currents in the absence of 2-APB, and was plotted as function of the concentration of 2-APB ([2-APB]o ).

    Techniques: Expressing, Concentration Assay, Inhibition

    Effect of 2-APB on LTP induced by two successive short tetani. Summarized time-course data for LTP in S-EPSP ( A ) or A-PS ( B ) induced by two successive tetani of 10 pulses (T 10 ) and 15 pulses (T 15 ), given in the absence (Control, empty circles) or the

    Journal:

    Article Title: Involvement of IP3 receptors in LTP and LTD induction in guinea pig hippocampal CA1 neurons

    doi: 10.1101/lm.17405

    Figure Lengend Snippet: Effect of 2-APB on LTP induced by two successive short tetani. Summarized time-course data for LTP in S-EPSP ( A ) or A-PS ( B ) induced by two successive tetani of 10 pulses (T 10 ) and 15 pulses (T 15 ), given in the absence (Control, empty circles) or the

    Article Snippet: A standard tetanus (100 pulses at 100 Hz) given in the presence of 2-APB induced a similar LTP to that seen in the absence of 2-APB, both being maintained for at least 60 min ( ).

    Techniques:

    Effects of 10 μM 2-APB on LTD. Summarized time-course plot for the S-EPSP ( A ) or A-PS ( B ) showing that LTD was induced by delivery of short LFSs (200 pulses at 1 Hz, three times at 18-min intervals; vertical bars) in standard solution (empty circles

    Journal:

    Article Title: Involvement of IP3 receptors in LTP and LTD induction in guinea pig hippocampal CA1 neurons

    doi: 10.1101/lm.17405

    Figure Lengend Snippet: Effects of 10 μM 2-APB on LTD. Summarized time-course plot for the S-EPSP ( A ) or A-PS ( B ) showing that LTD was induced by delivery of short LFSs (200 pulses at 1 Hz, three times at 18-min intervals; vertical bars) in standard solution (empty circles

    Article Snippet: A standard tetanus (100 pulses at 100 Hz) given in the presence of 2-APB induced a similar LTP to that seen in the absence of 2-APB, both being maintained for at least 60 min ( ).

    Techniques:

    Effects of 2-APB or MCPG on LTP induced by a standard tetanus of 100 pulses at 100 Hz (T 100 ). Summarized time-course plots for the S-EPSP ( A ) or A-PS ( B ) showing that LTP was induced by a standard tetanus applied in standard solution (empty circles) or

    Journal:

    Article Title: Involvement of IP3 receptors in LTP and LTD induction in guinea pig hippocampal CA1 neurons

    doi: 10.1101/lm.17405

    Figure Lengend Snippet: Effects of 2-APB or MCPG on LTP induced by a standard tetanus of 100 pulses at 100 Hz (T 100 ). Summarized time-course plots for the S-EPSP ( A ) or A-PS ( B ) showing that LTP was induced by a standard tetanus applied in standard solution (empty circles) or

    Article Snippet: A standard tetanus (100 pulses at 100 Hz) given in the presence of 2-APB induced a similar LTP to that seen in the absence of 2-APB, both being maintained for at least 60 min ( ).

    Techniques:

    Effects of 10 μM 2-APB and 50 μM AP5 on NMDAR-mediated synaptic responses. ( A ) Typical example of the time-course for NMDAR responses. The sample traces ( a–c ) were recorded at the times indicated. ( B ) Summarized time-course plot

    Journal:

    Article Title: Involvement of IP3 receptors in LTP and LTD induction in guinea pig hippocampal CA1 neurons

    doi: 10.1101/lm.17405

    Figure Lengend Snippet: Effects of 10 μM 2-APB and 50 μM AP5 on NMDAR-mediated synaptic responses. ( A ) Typical example of the time-course for NMDAR responses. The sample traces ( a–c ) were recorded at the times indicated. ( B ) Summarized time-course plot

    Article Snippet: A standard tetanus (100 pulses at 100 Hz) given in the presence of 2-APB induced a similar LTP to that seen in the absence of 2-APB, both being maintained for at least 60 min ( ).

    Techniques:

    Effect of 2-APB on synaptic responses elicited by test synaptic inputs and on LTPs induced by two successive tetani. ( A ) Summarized results for the S-EPSP (empty circles, n = 6) or A-PS (filled squares, n = 6) produced by 10-min application of 10 μM

    Journal:

    Article Title: Involvement of IP3 receptors in LTP and LTD induction in guinea pig hippocampal CA1 neurons

    doi: 10.1101/lm.17405

    Figure Lengend Snippet: Effect of 2-APB on synaptic responses elicited by test synaptic inputs and on LTPs induced by two successive tetani. ( A ) Summarized results for the S-EPSP (empty circles, n = 6) or A-PS (filled squares, n = 6) produced by 10-min application of 10 μM

    Article Snippet: A standard tetanus (100 pulses at 100 Hz) given in the presence of 2-APB induced a similar LTP to that seen in the absence of 2-APB, both being maintained for at least 60 min ( ).

    Techniques: Produced

    Abolition of fasiglifam-induced increase in nonselective cation channel (NSCC) current by inhibitors of the phospholipase (PLC)/protein kinase C (PKC) and TRP channels. ( a–c ) Effects of the nonselective transient receptor potential (TRP) channel blocker 2-aminoethyl diphenylborinate (2-APB; 10 μM) ( a ), TRP canonical (TRPC) channel blocker 3,5-bis(trifluoromethyl)pyrazole derivative 2 (BTP2; 10 μM) ( b ) and selective TRPC3 channel blocker pyrazole-3 (Pyr3; 10 μM) ( c ) on the NSCC current. ( d–f ) Effects of the PLC inhibitor U73122 (2 μM) ( d ) and PKC inhibitors Gö6983 (1 μM) ( e ) and Gö6976 (1 μM) ( f ) on the NSCC current. Fasiglifam-induced current increases were inhibited by these inhibitors. Rat single beta cells were voltage-clamped at −70 mV in the presence or absence of 10 μM fasiglifam, under the condition of 5.6 mM glucose and 100 μM tolbutamide throughout the experiments. The number of data points was five.

    Journal: Scientific Reports

    Article Title: Potentiation of Glucose-stimulated Insulin Secretion by the GPR40–PLC–TRPC Pathway in Pancreatic β-Cells

    doi: 10.1038/srep25912

    Figure Lengend Snippet: Abolition of fasiglifam-induced increase in nonselective cation channel (NSCC) current by inhibitors of the phospholipase (PLC)/protein kinase C (PKC) and TRP channels. ( a–c ) Effects of the nonselective transient receptor potential (TRP) channel blocker 2-aminoethyl diphenylborinate (2-APB; 10 μM) ( a ), TRP canonical (TRPC) channel blocker 3,5-bis(trifluoromethyl)pyrazole derivative 2 (BTP2; 10 μM) ( b ) and selective TRPC3 channel blocker pyrazole-3 (Pyr3; 10 μM) ( c ) on the NSCC current. ( d–f ) Effects of the PLC inhibitor U73122 (2 μM) ( d ) and PKC inhibitors Gö6983 (1 μM) ( e ) and Gö6976 (1 μM) ( f ) on the NSCC current. Fasiglifam-induced current increases were inhibited by these inhibitors. Rat single beta cells were voltage-clamped at −70 mV in the presence or absence of 10 μM fasiglifam, under the condition of 5.6 mM glucose and 100 μM tolbutamide throughout the experiments. The number of data points was five.

    Article Snippet: 2-APB (10 μM; Wako), BTP2, and Pyr3 were used as TRP channel blockers.

    Techniques: Planar Chromatography

    Effect of Ca 2+ channel inhibitors on calpain activity and T cell migration. Effect of ion channel inhibitors 2-APB (50 µM), SKF-96365 (100 µM), LaCl 3 (2 mM) on the following ( A–D ): ( A ) Calpain activity as detected by expression of calpain substrate CMAC, t -BOC-Leu-Met. Pseudo-color glow scale (red, highest to blue, lowest activity). Outline of DIC image shown as a white tracing. Scale bar = 10 µm. Images are representative of n = 4 experiments; ( B ) Individual T cells show random migration following observation for 20 min (n = 15 cells, n = 3 experiments); ( C ) Overall speed of T cell migration (n = 15 cells). *** P

    Journal: PLoS ONE

    Article Title: Calpain 2 Controls Turnover of LFA-1 Adhesions on Migrating T Lymphocytes

    doi: 10.1371/journal.pone.0015090

    Figure Lengend Snippet: Effect of Ca 2+ channel inhibitors on calpain activity and T cell migration. Effect of ion channel inhibitors 2-APB (50 µM), SKF-96365 (100 µM), LaCl 3 (2 mM) on the following ( A–D ): ( A ) Calpain activity as detected by expression of calpain substrate CMAC, t -BOC-Leu-Met. Pseudo-color glow scale (red, highest to blue, lowest activity). Outline of DIC image shown as a white tracing. Scale bar = 10 µm. Images are representative of n = 4 experiments; ( B ) Individual T cells show random migration following observation for 20 min (n = 15 cells, n = 3 experiments); ( C ) Overall speed of T cell migration (n = 15 cells). *** P

    Article Snippet: The following reagents were purchased as indicated: 2-APB, Merck Bioscience; lanthanum chloride (LaCl3 ), Sigma; SKF-96365, Sigma; pre-designed siRNAs for calpain 1 (ID 146578), calpain 2 (ID 112796 and 145947), and siRNA control #2 were all from Ambion; CAPN1 13 and 10, CAPN2 1 and 6 and negative control were from Qiagen.

    Techniques: Activity Assay, Migration, Expressing

    Knockdown of STIM1 attenuates SOCE in human NPCs. (A,B) Ca 2+ -responses during ER-store release and SOCE induced by Thapsigargin (TG, 10 μM) measured using the ratiometric Ca 2+ -indicator indo-1-AM in wild-type (WT) hNPCs (A) or hNPCs treated with pharmacological inhibitors of SOCE, BTP-2 and 2-APB at the indicated concentrations or DMSO as a solvent control (B) . Each trace represents the mean ±SEM for 25–100 cells. Ionomycin (Iono, 10 μM) was added at the end of each imaging to determine the peak F405/485 ratio obtained after saturation of the Ca 2+ -indicator with Ca 2+ . (C) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated treatment conditions. Mann–Whitney U test with Bonferroni correction. p = 1.819 × 10 -23 for DMSO control compared to BTP-2 treatment and p = 1.442 × 10 -45 for DMSO control compared to 2-APB treatment. (D) (Top) A representative Western blot showing levels of STIM1 protein in hNPCs transduced with an NTC (non-targeting control) or an sh-RNA targeting STIM1 ( STIM1 KD). Actin serves as the loading control. (Bottom) Quantification of STIM1 band intensities normalized to the loading control Actin from three independent biological replicates ( p = 0.00069, Student’s t -test). (E) Ca 2+ -responses during store-release and SOCE in hNPCs transduced with NTC and STIM1 KD. (F) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated genotypes. Peak F405/485 for store-release were not significantly different between NTC and STIM1 KD NPCs. p = 0.0001 for peak F405/485 during SOCE compared between NTC- and STIM1 KD NPCs. (G) Quantification of basal cytosolic [Ca 2+ ] values using Fura-2-AM in NTC- and STIM1 KD NPCs ( p = 1.115 × 10 -8 . Mann–Whitney U test. ( ∗∗∗ Indicates p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Stable STIM1 Knockdown in Self-Renewing Human Neural Precursors Promotes Premature Neural Differentiation

    doi: 10.3389/fnmol.2018.00178

    Figure Lengend Snippet: Knockdown of STIM1 attenuates SOCE in human NPCs. (A,B) Ca 2+ -responses during ER-store release and SOCE induced by Thapsigargin (TG, 10 μM) measured using the ratiometric Ca 2+ -indicator indo-1-AM in wild-type (WT) hNPCs (A) or hNPCs treated with pharmacological inhibitors of SOCE, BTP-2 and 2-APB at the indicated concentrations or DMSO as a solvent control (B) . Each trace represents the mean ±SEM for 25–100 cells. Ionomycin (Iono, 10 μM) was added at the end of each imaging to determine the peak F405/485 ratio obtained after saturation of the Ca 2+ -indicator with Ca 2+ . (C) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated treatment conditions. Mann–Whitney U test with Bonferroni correction. p = 1.819 × 10 -23 for DMSO control compared to BTP-2 treatment and p = 1.442 × 10 -45 for DMSO control compared to 2-APB treatment. (D) (Top) A representative Western blot showing levels of STIM1 protein in hNPCs transduced with an NTC (non-targeting control) or an sh-RNA targeting STIM1 ( STIM1 KD). Actin serves as the loading control. (Bottom) Quantification of STIM1 band intensities normalized to the loading control Actin from three independent biological replicates ( p = 0.00069, Student’s t -test). (E) Ca 2+ -responses during store-release and SOCE in hNPCs transduced with NTC and STIM1 KD. (F) Box plots quantifying the peak F405/485 values for store-release and SOCE in the indicated genotypes. Peak F405/485 for store-release were not significantly different between NTC and STIM1 KD NPCs. p = 0.0001 for peak F405/485 during SOCE compared between NTC- and STIM1 KD NPCs. (G) Quantification of basal cytosolic [Ca 2+ ] values using Fura-2-AM in NTC- and STIM1 KD NPCs ( p = 1.115 × 10 -8 . Mann–Whitney U test. ( ∗∗∗ Indicates p

    Article Snippet: To check for inhibition of SOCE, inhibitors of the SOC-channel, BTP-2 (Invitrogen) and 2-APB (Invitrogen) or DMSO (solvent control) were dissolved in the indicated concentration in 100 μl of the ‘0 Ca2+ HBSS.’ For these experiments, the culture medium in which the cells were kept after washing was replaced by HBSS containing either the inhibitors or DMSO and imaged.

    Techniques: Imaging, MANN-WHITNEY, Western Blot, Transduction