k252a  (Alomone Labs)


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    Structured Review

    Alomone Labs k252a
    A – D , Effect of <t>K252a</t> and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    K252a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells"

    Article Title: Sorting of Internalized Neurotrophins into an Endocytic Transcytosis Pathway via the Golgi System: Ultrastructural Analysis in Retinal Ganglion Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-22-08915.2001

    A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.
    Figure Legend Snippet: A – D , Effect of K252a and trkC activity on the distribution of internalized neurotrophins in retinal ganglion cells ( RGC ) ( A – C ) and SDS-PAGE analysis of internalized neurotrophins in purified RGCs ( D ). A , Labeling densities of internalized BDNF and NT-3 in the Golgi system of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. B , Labeling densities of internalized BDNF and NT-3 in lysosomes and endosomes of RGCs with and without K252a. The analysis was done in triplicate; significance was determined by unpaired t test. C , Quantification of anterograde transport of 50–80 ng radiolabeled NGF when coinjected in the eye with 50–60 ng cold NT-3 or BDNF. The number of experiments is indicated. Significance was determined by unpaired t test. Error bars indicate SEM. D , SDS-PAGE (15%) of internalized neurotrophins NGF, BDNF, and NT-3 recovered from purified immunopanned RGCs after 10 hr. Each sample was run with an adjacent sample of native same factor ( Na ). The molecular weight is indicated. Arrow indicates the dye front. Note that much of the BDNF recovered from RGCs is cleaved, whereas virtually all the NGF and NT-3 is intact protein by this analysis.

    Techniques Used: Activity Assay, SDS Page, Purification, Labeling, Molecular Weight

    Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.
    Figure Legend Snippet: Internalization of 125 I-NT-3 (10–20 ng/ml) in purified retinal ganglion cells from E18–21 chick embryos does not require tyrosine kinase activity. The amount of internalization in the presence of 1 μg/ml K252a (tyrosine kinase inhibitor) is plotted as the percentage relative to the values for vehicle (1 μg/ml DMSO), which were averaged to 100% internalization (∼40,000 cpm per plate). Nonspecific association of NT-3 (at 4°C) is indicated ( dotted line ). Error bars indicate SEM. The number of independent experiments (each in duplicate) is indicated. The p level for confidence ( t test) is indicated, showing no significant effect of K252a on NT-3 internalization.

    Techniques Used: Purification, Activity Assay

    Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).
    Figure Legend Snippet: Diagram summarizing proposed subcellular pathways of internalized NT-3 ( black dots ) in a retinal ganglion cell. At least two pathways of internalized NT-3 can be distinguished. A lysosomal pathway of NT-3 may be common to all neurotrophins and may involve binding to the p75 receptor ( U ) or binding of NT-3 to trkC receptor ( Y ) in the presence of the tyrosine kinase inhibitor K252a. The neurotrophin is degraded in lysosomes ( LYS ). Alternatively, a novel pathway of NT-3 internalized in endosomes fuses with membranes of the Golgi apparatus: Golgi Pathway . This sorting requires tyrosine kinase activity (presumably trkC , Y ), and this pathway may join that of newly synthesized neurotrophins as well as p75 receptor ( U ) from the endoplasmic reticulum ( ER ). After passage through the Golgi system, internalized NT-3 is packaged in presumptive large dense-core vesicles ( LDCV ) for anterograde axonal transport. In this pathway, internalized NT-3 binds preferentially to p75. Anterogradely transported NT-3 is released from RGC axon terminals in the tectum, and after release it binds predominantly to trkC in tectal cells where it accumulates in multivesicular bodies ( MVB ).

    Techniques Used: Binding Assay, Activity Assay, Synthesized

    A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.
    Figure Legend Snippet: A – C , Anterograde axonal transport of neurotrophic factors and cytochrome c in chick RGCs. A , B , The relative amount of anterograde transport of radiolabeled NT-3 was plotted by dividing the amount measured by gamma counting in the tectum (counts per minute per specific activity in picograms) by the amount measured in the eye (counts per minute per specific activity in nanograms) at the time chick embryos were killed (20 hr after injection in the eye). A , The effects of coinjection of monensin ( MON ), excess cold NT-3 ( cold NT-3 ), excess cold NGF ( cold NGF ), or excess cold BDNF ( cold BDNF ), blocking p75 antibody ( aP75 ), blocking trkC antibody ( atrkC ), and normal rabbit IgG ( IgG ). B , The effects of coinjection of monensin ( MON ), tyrosine kinase inhibitor K252a ( K252a ), vehicle ( DMSO ), wortmannin ( WOR ), LY294,002 ( LY ), and brefeldin A ( BFA ) are indicated. C , The relative amount of anterograde transport of radiolabeled glial cell line-derived neurotrophic factor ( GDNF ), cardiotrophin-1 ( CT1 ), and cytochrome c ( CytC ) was plotted by dividing the amount measured by gamma counting in the midbrain (picograms) by the amount measured in the eye (nanograms) at the time chick embryos were killed (20 hr after injection in the eye). The effects of coinjection of monensin ( MON ), excess cold GDNF, or CytC, and K252a are indicated. Error bars indicate SEM. The number of independent experiments is indicated. *** p ≤ 0.005; ** p ≤ 0.01; * p ≤ 0.05.

    Techniques Used: Activity Assay, Injection, Blocking Assay, Derivative Assay

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    Alomone Labs kv1 5 c terminus
    Binding of the C-terminal domain of Kv to Kvβ (a)  Western blots of Kvβ2 (upper panel) and Kvβ3 (middle panel) pulled down by the GST-Kv1.5 C-terminus fusion peptides. Fusion proteins containing 60, 38, or 19 terminal amino acid peptides from Kvα1.5 C-terminus attached to GST or GST with unrelated peptide (Control; 30μ g each) were incubated with lysate of Kvβ2 or Kvβ3 -expressing  E.coli  (350 μ g total protein). Protein complexes were pulled down using GST·Bind beads, washed and eluted with 10mM glutathione. The eluate was separated by SDS-PAGE and probed with anti-pan-Kvβ antibody, an antibody directed against the C-terminus of Kv1.5 (bait) or GST;  (b)  Densitometric analysis of the bands in panel a. The density of the Kvβ band precipitated with GST-C60 was assigned a 100% value. †, P
    Kv1 5 C Terminus, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Alomone Labs ω conotoxin mviic mviic
    The effects of nifedipine, <t>ω-conotoxin</t> GVIA, ω-conotoxin <t>MVIIC</t> with ω-agatoxin TK and CdCl 2 on Ba 2+ currents In panels A , B , C and D left plots present data from the newborn and right plots from the adult animals. A , Ba 2+ currents were evoked during 30 ms voltage steps from −80 mV to +60 mV. All recordings were obtained after preincubation (2 min) with different VACC blockers as indicated. B , current densities from the same cells were plotted to obtain the Ba 2+ current I–V relationship. C , the effect of VACC blockers on the Ba 2+ current I–V relationship obtained from 300 ms voltage ramps. D , the time course of the effect of nifedipine (1), GVIA (2), MVIIC/TK (3) and CdCl 2 (4) on Ba 2+ currents. LVA currents in adults are shown as red triangles and the HVA are shown as black triangles. Note that current values were normalized to the peak amplitude of both VACC components. Nifedipine blocked about 50% of the LVA component; however, other toxins than Cd 2+ did not affect the LVA component.
    ω Conotoxin Mviic Mviic, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Binding of the C-terminal domain of Kv to Kvβ (a)  Western blots of Kvβ2 (upper panel) and Kvβ3 (middle panel) pulled down by the GST-Kv1.5 C-terminus fusion peptides. Fusion proteins containing 60, 38, or 19 terminal amino acid peptides from Kvα1.5 C-terminus attached to GST or GST with unrelated peptide (Control; 30μ g each) were incubated with lysate of Kvβ2 or Kvβ3 -expressing  E.coli  (350 μ g total protein). Protein complexes were pulled down using GST·Bind beads, washed and eluted with 10mM glutathione. The eluate was separated by SDS-PAGE and probed with anti-pan-Kvβ antibody, an antibody directed against the C-terminus of Kv1.5 (bait) or GST;  (b)  Densitometric analysis of the bands in panel a. The density of the Kvβ band precipitated with GST-C60 was assigned a 100% value. †, P

    Journal: Pflugers Archiv

    Article Title: Interactions between the C-terminus of Kv1.5 and Kv? regulate pyridine nucleotide-dependent changes in channel gating

    doi: 10.1007/s00424-012-1093-z

    Figure Lengend Snippet: Binding of the C-terminal domain of Kv to Kvβ (a) Western blots of Kvβ2 (upper panel) and Kvβ3 (middle panel) pulled down by the GST-Kv1.5 C-terminus fusion peptides. Fusion proteins containing 60, 38, or 19 terminal amino acid peptides from Kvα1.5 C-terminus attached to GST or GST with unrelated peptide (Control; 30μ g each) were incubated with lysate of Kvβ2 or Kvβ3 -expressing E.coli (350 μ g total protein). Protein complexes were pulled down using GST·Bind beads, washed and eluted with 10mM glutathione. The eluate was separated by SDS-PAGE and probed with anti-pan-Kvβ antibody, an antibody directed against the C-terminus of Kv1.5 (bait) or GST; (b) Densitometric analysis of the bands in panel a. The density of the Kvβ band precipitated with GST-C60 was assigned a 100% value. †, P

    Article Snippet: Antibodies were obtained from the following sources: pan-Kvβ and Kv1.5 N-terminus (Santa-Cruz Biotechnologies), Kv1.5 C-terminus (Alomone Labs), Kvβ2, β1.1, β1.2 (Neuromab), GST (Novagen).

    Techniques: Binding Assay, Western Blot, Incubation, Expressing, SDS Page

    The effects of nifedipine, ω-conotoxin GVIA, ω-conotoxin MVIIC with ω-agatoxin TK and CdCl 2 on Ba 2+ currents In panels A , B , C and D left plots present data from the newborn and right plots from the adult animals. A , Ba 2+ currents were evoked during 30 ms voltage steps from −80 mV to +60 mV. All recordings were obtained after preincubation (2 min) with different VACC blockers as indicated. B , current densities from the same cells were plotted to obtain the Ba 2+ current I–V relationship. C , the effect of VACC blockers on the Ba 2+ current I–V relationship obtained from 300 ms voltage ramps. D , the time course of the effect of nifedipine (1), GVIA (2), MVIIC/TK (3) and CdCl 2 (4) on Ba 2+ currents. LVA currents in adults are shown as red triangles and the HVA are shown as black triangles. Note that current values were normalized to the peak amplitude of both VACC components. Nifedipine blocked about 50% of the LVA component; however, other toxins than Cd 2+ did not affect the LVA component.

    Journal: The Journal of Physiology

    Article Title: Voltage-activated Ca2+ channels and their role in the endocrine function of the pituitary gland in newborn and adult mice

    doi: 10.1113/jphysiol.2003.058271

    Figure Lengend Snippet: The effects of nifedipine, ω-conotoxin GVIA, ω-conotoxin MVIIC with ω-agatoxin TK and CdCl 2 on Ba 2+ currents In panels A , B , C and D left plots present data from the newborn and right plots from the adult animals. A , Ba 2+ currents were evoked during 30 ms voltage steps from −80 mV to +60 mV. All recordings were obtained after preincubation (2 min) with different VACC blockers as indicated. B , current densities from the same cells were plotted to obtain the Ba 2+ current I–V relationship. C , the effect of VACC blockers on the Ba 2+ current I–V relationship obtained from 300 ms voltage ramps. D , the time course of the effect of nifedipine (1), GVIA (2), MVIIC/TK (3) and CdCl 2 (4) on Ba 2+ currents. LVA currents in adults are shown as red triangles and the HVA are shown as black triangles. Note that current values were normalized to the peak amplitude of both VACC components. Nifedipine blocked about 50% of the LVA component; however, other toxins than Cd 2+ did not affect the LVA component.

    Article Snippet: Purified ω-conotoxin GVIA (GVIA), ω-conotoxin MVIIC (MVIIC), ω-agatoxin TK (TK) and SNX-482 were purchased from Alomone Laboratories (Jerusalem, Israel) and dissolved in distilled water to obtain stock solutions at concentrations of 100 μ m , 10 μ m , 1 μ m and 1 μ m , respectively.

    Techniques: Mass Spectrometry

    Ca V 2.1/2.2 contribute to AIS calcium in the somatosensory cortex. A. Representative effect of ω-conotoxin MVIIC application on AIS calcium in L5b pyramidal cells in the somatosensory cortex. Left: example time-locked control cell. Black, baseline; gray, post. Right: example of the effect of ω-conotoxin MVIIC. Black, baseline; green, ω-conotoxin MVIIC. Linescan data are plotted as mean ± standard error. B. Summary of the effects of ω-conotoxin MVIIC on AIS calcium in somatosensory L5b pyramidal neurons. Black, time-locked control cells; green, ω-conotoxin MVIIC. C. Decreases in EPSP amplitude confirm the presence of ω-conotoxin MVIIC at the slice. Top: representative examples ofPSPs in ω-conotoxin MVIIC (right) or in time-locked control cells (left). Bottom: Summary of the effects of ω-conotoxin MVIIC on EPSP amplitude in somatosensory cortex. Black, baseline; gray, time-locked control; green, ω-conotoxin MVIIC.

    Journal: bioRxiv

    Article Title: Functional microstructure of CaV-mediated calcium signaling in the axon initial segment

    doi: 10.1101/2020.11.13.382374

    Figure Lengend Snippet: Ca V 2.1/2.2 contribute to AIS calcium in the somatosensory cortex. A. Representative effect of ω-conotoxin MVIIC application on AIS calcium in L5b pyramidal cells in the somatosensory cortex. Left: example time-locked control cell. Black, baseline; gray, post. Right: example of the effect of ω-conotoxin MVIIC. Black, baseline; green, ω-conotoxin MVIIC. Linescan data are plotted as mean ± standard error. B. Summary of the effects of ω-conotoxin MVIIC on AIS calcium in somatosensory L5b pyramidal neurons. Black, time-locked control cells; green, ω-conotoxin MVIIC. C. Decreases in EPSP amplitude confirm the presence of ω-conotoxin MVIIC at the slice. Top: representative examples ofPSPs in ω-conotoxin MVIIC (right) or in time-locked control cells (left). Bottom: Summary of the effects of ω-conotoxin MVIIC on EPSP amplitude in somatosensory cortex. Black, baseline; gray, time-locked control; green, ω-conotoxin MVIIC.

    Article Snippet: Chemicals TTA-P2 was from Alomone Labs. ω-conotoxin-MVIIC, ω-conotoxin-GVIA, ω-agatoxin-TK, and SNX-482 were from Peptides International.

    Techniques:

    Ca V 3 channels and Ca V 2.1/2.2 exhibit distinct functional distributions. A. Schematic of 2PLSM point scan imaging protocol. Points were imaged in sets of 5, with each point separated by 0.5 µm. The laser was parked at a single diffraction-limited point for 25 ms preceding and 100 ms following an AP and calcium influx was measured with OGB-5N. Points were scanned in the sequence 2, 4, 1, 3, 5 and each point sampled a single AP. Data was averaged over 20-50 repetitions. B. Isochronal calcium peaks from neighboring point sets. Calcium influx at each point is color-coded as in panel A. B1 shows a point set with a hotspot at point 5. B2 is the point set immediately adjacent to B1 and shows equivalent calcium influx across all points. Gray bar indicates the calcium transient onset and offset. C. Distribution of point sets containing hotspots. Peak calcium influx at the brightest point was divided by the isochronal calcium influx at the point(s) 1 µm away. 46 of 59 sites imaged fell within a normal distribution, while 13 sites exhibited higher relative calcium influx. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Red line indicates the fit of a normal distribution. Total distribution fit for normality (Shapiro Wilk test p = 0.0016). D. Calcium influx at hotspots was approximately 2x higher than calcium influx at non-hotspot points. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± standard deviation. E. Comparison of the flanks of point sets with a local hotspot and those without. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± standard deviation for each 0.5 µm increment from the brightest point of the set. F.Influence of selective Ca V antagonists or store depletion on peak calcium influx during point scan imaging. Circles represent single point sets. Black, control; pink, TTA-P2; green, ω-conotoxin MVIIC; orange, CPA. G. Influence of selective Ca V antagonists or store depletion on calcium hotspots. Hotspots were classified as points > 1.5 times brighter than the point(s) 1 µm away. Dotted gray line represents the distinction between point sets with a local hotspot (above) and those without (below). Color code as in panel F.

    Journal: bioRxiv

    Article Title: Functional microstructure of CaV-mediated calcium signaling in the axon initial segment

    doi: 10.1101/2020.11.13.382374

    Figure Lengend Snippet: Ca V 3 channels and Ca V 2.1/2.2 exhibit distinct functional distributions. A. Schematic of 2PLSM point scan imaging protocol. Points were imaged in sets of 5, with each point separated by 0.5 µm. The laser was parked at a single diffraction-limited point for 25 ms preceding and 100 ms following an AP and calcium influx was measured with OGB-5N. Points were scanned in the sequence 2, 4, 1, 3, 5 and each point sampled a single AP. Data was averaged over 20-50 repetitions. B. Isochronal calcium peaks from neighboring point sets. Calcium influx at each point is color-coded as in panel A. B1 shows a point set with a hotspot at point 5. B2 is the point set immediately adjacent to B1 and shows equivalent calcium influx across all points. Gray bar indicates the calcium transient onset and offset. C. Distribution of point sets containing hotspots. Peak calcium influx at the brightest point was divided by the isochronal calcium influx at the point(s) 1 µm away. 46 of 59 sites imaged fell within a normal distribution, while 13 sites exhibited higher relative calcium influx. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Red line indicates the fit of a normal distribution. Total distribution fit for normality (Shapiro Wilk test p = 0.0016). D. Calcium influx at hotspots was approximately 2x higher than calcium influx at non-hotspot points. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± standard deviation. E. Comparison of the flanks of point sets with a local hotspot and those without. Black, point sets containing a local hotspot; gray, point sets with no local hotspot. Data are plotted as mean ± standard deviation for each 0.5 µm increment from the brightest point of the set. F.Influence of selective Ca V antagonists or store depletion on peak calcium influx during point scan imaging. Circles represent single point sets. Black, control; pink, TTA-P2; green, ω-conotoxin MVIIC; orange, CPA. G. Influence of selective Ca V antagonists or store depletion on calcium hotspots. Hotspots were classified as points > 1.5 times brighter than the point(s) 1 µm away. Dotted gray line represents the distinction between point sets with a local hotspot (above) and those without (below). Color code as in panel F.

    Article Snippet: Chemicals TTA-P2 was from Alomone Labs. ω-conotoxin-MVIIC, ω-conotoxin-GVIA, ω-agatoxin-TK, and SNX-482 were from Peptides International.

    Techniques: Functional Assay, Imaging, Sequencing, Standard Deviation

    Sodium and calcium influx occur on the rising and falling phases of the AP at physiological temperatures, respectively. A. Pointscan imaging protocol was performed as in Figure 4A . OGB-5N was replaced with the sodium indicator ING-2, and Alexa-594 was excluded from the internal recording solution. B. Representative ING-2 sodium imaging pointset. Points are color-coded as in Panel A. Gray bar indicates the sodium transient onset and offset. C. Distribution of sodium imaging point sets calculated as in Fig 4C . Red line represents the fit of a normal distribution to the data. D. Sodium and calcium transients from pointscan imaging temporally-aligned to AP threshold and peak. Left: Sodium and calcium influx onset relative to AP threshold. Right: Sodium and calcium influx onset relative to AP peak. Transient onset time was measured for the brightest point in a point set. Circles are individual point sets. Gray dashed line shows AP threshold (left) or peak (right) timing. Red, ING-2 sodium imaging; black, OGB-5N calcium imaging in control conditions; blue, calcium imaging in the presence of TTA-P2; green, calcium imaging in the presence of ω-conotoxin MVIIC; orange, calcium imaging in the presence of cyclopiazonic acid.

    Journal: bioRxiv

    Article Title: Functional microstructure of CaV-mediated calcium signaling in the axon initial segment

    doi: 10.1101/2020.11.13.382374

    Figure Lengend Snippet: Sodium and calcium influx occur on the rising and falling phases of the AP at physiological temperatures, respectively. A. Pointscan imaging protocol was performed as in Figure 4A . OGB-5N was replaced with the sodium indicator ING-2, and Alexa-594 was excluded from the internal recording solution. B. Representative ING-2 sodium imaging pointset. Points are color-coded as in Panel A. Gray bar indicates the sodium transient onset and offset. C. Distribution of sodium imaging point sets calculated as in Fig 4C . Red line represents the fit of a normal distribution to the data. D. Sodium and calcium transients from pointscan imaging temporally-aligned to AP threshold and peak. Left: Sodium and calcium influx onset relative to AP threshold. Right: Sodium and calcium influx onset relative to AP peak. Transient onset time was measured for the brightest point in a point set. Circles are individual point sets. Gray dashed line shows AP threshold (left) or peak (right) timing. Red, ING-2 sodium imaging; black, OGB-5N calcium imaging in control conditions; blue, calcium imaging in the presence of TTA-P2; green, calcium imaging in the presence of ω-conotoxin MVIIC; orange, calcium imaging in the presence of cyclopiazonic acid.

    Article Snippet: Chemicals TTA-P2 was from Alomone Labs. ω-conotoxin-MVIIC, ω-conotoxin-GVIA, ω-agatoxin-TK, and SNX-482 were from Peptides International.

    Techniques: Imaging

    Rescue of spontaneous synaptic activity in BoNT/A-silenced synapses by elevated extracellular Ca 2+ requires VGCCs. ( A ) Representative whole-cell recordings and mean mEPSC frequencies from non-intoxicated cultures in the presence of 2, 4, 8, or 16 mM Ca 2+ . Representative whole-cell recordings and mean mEPSC frequencies from BoNT/A-intoxicated cultures, in the presence of 2, 4, 8, or 16 mM Ca 2+ as well in 16 mM Ca 2+ with VGCC antagonists (10 µM nimodipine, 0.5 µM ω-agatoxin IVA and 0.5 µM ω-conotoxin MVIIC). ( C ) Representative whole-cell recordings and mean mEPSC frequencies from neurons intoxicated by BoNT/D or BoNT/E and incubated in 2 or 16 mM Ca 2+ . mEPSC frequencies are normalized to recordings from age-matched, non-intoxicated control cultures. Scale bars represent 5 s (x-axis) and 40 pA (y-axis). All data presented as mean ± SEM and n ≥ 10 neurons for all conditions. **Indicates p

    Journal: Scientific Reports

    Article Title: Use-dependent potentiation of voltage-gated calcium channels rescues neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A

    doi: 10.1038/s41598-017-16064-3

    Figure Lengend Snippet: Rescue of spontaneous synaptic activity in BoNT/A-silenced synapses by elevated extracellular Ca 2+ requires VGCCs. ( A ) Representative whole-cell recordings and mean mEPSC frequencies from non-intoxicated cultures in the presence of 2, 4, 8, or 16 mM Ca 2+ . Representative whole-cell recordings and mean mEPSC frequencies from BoNT/A-intoxicated cultures, in the presence of 2, 4, 8, or 16 mM Ca 2+ as well in 16 mM Ca 2+ with VGCC antagonists (10 µM nimodipine, 0.5 µM ω-agatoxin IVA and 0.5 µM ω-conotoxin MVIIC). ( C ) Representative whole-cell recordings and mean mEPSC frequencies from neurons intoxicated by BoNT/D or BoNT/E and incubated in 2 or 16 mM Ca 2+ . mEPSC frequencies are normalized to recordings from age-matched, non-intoxicated control cultures. Scale bars represent 5 s (x-axis) and 40 pA (y-axis). All data presented as mean ± SEM and n ≥ 10 neurons for all conditions. **Indicates p

    Article Snippet: For BoNT/E, toxin was activated by a 60 min incubation at 37 °C with 0.3 mg/mL TPCK-treated trypsin in 0.05 M sodium phosphate buffer (pH 6.5) . ω-agatoxin IVA, ω-conotoxin MVIIC and GV-58 were purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Activity Assay, Incubation