anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    Induction of apoptosis without ROS production and its suppression by RasGRP2 without the Rap1 pathway. ( a , b , e , f ) Cell viability was determined by Cell Counting Kit-8 assay. Cells were incubated with or without BAM7 ( a ) or <t>anisomycin</t> ( b ) for 24 h to 48 h. Cell viability at 0 h was taken as 100%, circle: M control, square: BAM7- or anisomycin-treated M cells, triangle: R control, cross: BAM7- or anisomycin-treated R cells. ( c ) Apoptosis for 24 h was determined by NucView488; scale bar = 250 μm. ( d ) Apoptosis ratio in M cells control was taken as 1. ( e ) Cells were pre-incubated with or without NAC or DPI and incubated with or without BAM7 for 48 h. ( f ) Cells were pre-treated with siRNA against Rap1A or negative control siRNA and incubated with or without BAM7 or TNF-α for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control at the same time point, and ## P < 0.01 compared with each R cell transfected with the negative control siRNA.
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells"

    Article Title: The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-53419-4

    Induction of apoptosis without ROS production and its suppression by RasGRP2 without the Rap1 pathway. ( a , b , e , f ) Cell viability was determined by Cell Counting Kit-8 assay. Cells were incubated with or without BAM7 ( a ) or anisomycin ( b ) for 24 h to 48 h. Cell viability at 0 h was taken as 100%, circle: M control, square: BAM7- or anisomycin-treated M cells, triangle: R control, cross: BAM7- or anisomycin-treated R cells. ( c ) Apoptosis for 24 h was determined by NucView488; scale bar = 250 μm. ( d ) Apoptosis ratio in M cells control was taken as 1. ( e ) Cells were pre-incubated with or without NAC or DPI and incubated with or without BAM7 for 48 h. ( f ) Cells were pre-treated with siRNA against Rap1A or negative control siRNA and incubated with or without BAM7 or TNF-α for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control at the same time point, and ## P < 0.01 compared with each R cell transfected with the negative control siRNA.
    Figure Legend Snippet: Induction of apoptosis without ROS production and its suppression by RasGRP2 without the Rap1 pathway. ( a , b , e , f ) Cell viability was determined by Cell Counting Kit-8 assay. Cells were incubated with or without BAM7 ( a ) or anisomycin ( b ) for 24 h to 48 h. Cell viability at 0 h was taken as 100%, circle: M control, square: BAM7- or anisomycin-treated M cells, triangle: R control, cross: BAM7- or anisomycin-treated R cells. ( c ) Apoptosis for 24 h was determined by NucView488; scale bar = 250 μm. ( d ) Apoptosis ratio in M cells control was taken as 1. ( e ) Cells were pre-incubated with or without NAC or DPI and incubated with or without BAM7 for 48 h. ( f ) Cells were pre-treated with siRNA against Rap1A or negative control siRNA and incubated with or without BAM7 or TNF-α for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control at the same time point, and ## P < 0.01 compared with each R cell transfected with the negative control siRNA.

    Techniques Used: Cell Counting, Incubation, Negative Control, Over Expression, Transfection

    Activation of R-Ras-PI3K-Akt signaling pathway by RasGRP2 and suppression of apoptosis via its signaling pathway. ( a – e ) Each effect was detected by western blotting. ( a ) Activated R-Ras and Rap2A were isolated by pull-down assay using RalGDS-RBD agarose beads. ( b ) Activated TC21 was isolated by pull-down assay using Raf1-RBD agarose beads. ( c – e ) Phosphorylation of Akt was detected using specific antibodies. Cells were treated with siRNAs against R-Ras or negative control siRNA ( d ), or incubated with or without LY294002 ( e ). ( f , g ) Cell viability was determined by Cell Counting Kit-8 assay. ( f ) Cells were pre-treatment with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 or anisomycin for 48 h. ( g ) Cells were pre-incubated with or without LY294002 and incubated with or without BAM7 or anisomycin for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control, ## P < 0.01 compared with each R cell without pre-treatment or transfected with the negative control siRNA, and ++ P < 0.01 compared with each M cell transfected with the negative control siRNA.
    Figure Legend Snippet: Activation of R-Ras-PI3K-Akt signaling pathway by RasGRP2 and suppression of apoptosis via its signaling pathway. ( a – e ) Each effect was detected by western blotting. ( a ) Activated R-Ras and Rap2A were isolated by pull-down assay using RalGDS-RBD agarose beads. ( b ) Activated TC21 was isolated by pull-down assay using Raf1-RBD agarose beads. ( c – e ) Phosphorylation of Akt was detected using specific antibodies. Cells were treated with siRNAs against R-Ras or negative control siRNA ( d ), or incubated with or without LY294002 ( e ). ( f , g ) Cell viability was determined by Cell Counting Kit-8 assay. ( f ) Cells were pre-treatment with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 or anisomycin for 48 h. ( g ) Cells were pre-incubated with or without LY294002 and incubated with or without BAM7 or anisomycin for 48 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control, ## P < 0.01 compared with each R cell without pre-treatment or transfected with the negative control siRNA, and ++ P < 0.01 compared with each M cell transfected with the negative control siRNA.

    Techniques Used: Activation Assay, Western Blot, Isolation, Pull Down Assay, Negative Control, Incubation, Cell Counting, Over Expression, Transfection

    Inhibition of Bax translocation by RasGRP2 via R-Ras-PI3K-Akt signaling pathway. ( a – d ) Each effect was detected by western blotting. ( a ) Phosphorylation of JNK was detected using a specific antibody. Cells were incubated with or without TNF-α, anisomycin, or BAM7 for 1 h. ( b ) Activated Bax was isolated by immunoprecipitation (IP) using a Bax (6A7) antibody. Cells were incubated with or without BAM7 or anisomycin for 16 h. ( c , d ) Cytosolic and mitochondrial fractions were isolated by cell fractionation kit. ( d ) Cells were pre-treated with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 for 16 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin, IP: immunoprecipitation.
    Figure Legend Snippet: Inhibition of Bax translocation by RasGRP2 via R-Ras-PI3K-Akt signaling pathway. ( a – d ) Each effect was detected by western blotting. ( a ) Phosphorylation of JNK was detected using a specific antibody. Cells were incubated with or without TNF-α, anisomycin, or BAM7 for 1 h. ( b ) Activated Bax was isolated by immunoprecipitation (IP) using a Bax (6A7) antibody. Cells were incubated with or without BAM7 or anisomycin for 16 h. ( c , d ) Cytosolic and mitochondrial fractions were isolated by cell fractionation kit. ( d ) Cells were pre-treated with siRNAs against R-Ras or negative control siRNA and incubated with or without BAM7 for 16 h. M: mock cells, R: RasGRP2-stable overexpression cells, aniso: anisomycin, IP: immunoprecipitation.

    Techniques Used: Inhibition, Translocation Assay, Western Blot, Incubation, Isolation, Immunoprecipitation, Cell Fractionation, Negative Control, Over Expression

    Suppression of apoptosis by promoting mitochondrial hexokinase-2 translocation via RasGRP2-R-Ras-PI3K-Akt signaling pathway. ( a – c ) Cells were pre-incubated with or without LY294002 ( b ) or CTZ ( c ) and incubated with or without BAM7 for 16 h. Each effect was detected by western blotting. Cytosol and mitochondrial fractions were isolated by cell fractionation kit. ( d ) Cells were pre-incubated with or without CTZ and incubated with or without BAM7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay. M: mock cells, R: RasGRP2-stable overexpression cells, CTZ: clotrimazole, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control, ## P < 0.01 compared with each R cell without pre-treatment, and ++ P < 0.01 compared with each M cell without pre-treatment.
    Figure Legend Snippet: Suppression of apoptosis by promoting mitochondrial hexokinase-2 translocation via RasGRP2-R-Ras-PI3K-Akt signaling pathway. ( a – c ) Cells were pre-incubated with or without LY294002 ( b ) or CTZ ( c ) and incubated with or without BAM7 for 16 h. Each effect was detected by western blotting. Cytosol and mitochondrial fractions were isolated by cell fractionation kit. ( d ) Cells were pre-incubated with or without CTZ and incubated with or without BAM7 for 48 h. Cell viability was determined by Cell Counting Kit-8 assay. M: mock cells, R: RasGRP2-stable overexpression cells, CTZ: clotrimazole, aniso: anisomycin. Data are shown as the mean ± SD (n = 3), ** P < 0.01 compared with each cell control, ## P < 0.01 compared with each R cell without pre-treatment, and ++ P < 0.01 compared with each M cell without pre-treatment.

    Techniques Used: Translocation Assay, Incubation, Western Blot, Isolation, Cell Fractionation, Cell Counting, Over Expression

    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using <t>anisomycin</t> increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anisomycin/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
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    anisomycin - by Bioz Stars, 2023-09
    94/100 stars

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    1) Product Images from "Neu3 Sialidase-Mediated Ganglioside Conversion Is Necessary for Axon Regeneration and Is Blocked in CNS Axons"

    Article Title: Neu3 Sialidase-Mediated Ganglioside Conversion Is Necessary for Axon Regeneration and Is Blocked in CNS Axons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4432-13.2014

    P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using anisomycin increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.
    Figure Legend Snippet: P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using anisomycin increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.

    Techniques Used: Activation Assay, Labeling, Activity Assay, Two Tailed Test

    P38MAPK and ERK are not activated within 15 min after axotomy in retinal axons, but forced activation of P38MAPK increases Neu3 sialidase activity in retinal axons. I, A–D, Fluorescent photomicrographs of adult retinal axons uncut (A,C) and 15 min after axotomy (B,D), double-labeled for P38MAPK (A,B) or ERK (C,D) (red) and pP38MAPK (A,B) or pERK (C,D) (green). Ratios of pP38MAPK/P38MAPK and pERK/ERK intensities (± SEM) normalized against background intensity levels are plotted in E and F (30–40 axons/chamber, n = 4 chambers). II, G, H, Fluorescent photomicrographs of adult uncut retinal axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red). Anisomycin increased the GM1/GD1a ratio (compare H with G). I, Ratios of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels are plotted in I (30–40 axons/chamber, n ≥ 7). ***p < 0.001 (Student's t test, 2-tailed). Scale bars, 50 μm.
    Figure Legend Snippet: P38MAPK and ERK are not activated within 15 min after axotomy in retinal axons, but forced activation of P38MAPK increases Neu3 sialidase activity in retinal axons. I, A–D, Fluorescent photomicrographs of adult retinal axons uncut (A,C) and 15 min after axotomy (B,D), double-labeled for P38MAPK (A,B) or ERK (C,D) (red) and pP38MAPK (A,B) or pERK (C,D) (green). Ratios of pP38MAPK/P38MAPK and pERK/ERK intensities (± SEM) normalized against background intensity levels are plotted in E and F (30–40 axons/chamber, n = 4 chambers). II, G, H, Fluorescent photomicrographs of adult uncut retinal axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red). Anisomycin increased the GM1/GD1a ratio (compare H with G). I, Ratios of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels are plotted in I (30–40 axons/chamber, n ≥ 7). ***p < 0.001 (Student's t test, 2-tailed). Scale bars, 50 μm.

    Techniques Used: Activation Assay, Activity Assay, Labeling

    anisomycin  (Alomone Labs)


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    Alomone Labs anisomycin
    P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using <t>anisomycin</t> increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.
    Anisomycin, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anisomycin/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anisomycin - by Bioz Stars, 2023-09
    94/100 stars

    Images

    1) Product Images from "Neu3 Sialidase-Mediated Ganglioside Conversion Is Necessary for Axon Regeneration and Is Blocked in CNS Axons"

    Article Title: Neu3 Sialidase-Mediated Ganglioside Conversion Is Necessary for Axon Regeneration and Is Blocked in CNS Axons

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4432-13.2014

    P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using anisomycin increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.
    Figure Legend Snippet: P38MAPK activation is the intermediary step between eCa2+ influx and Neu3 sialidase activation after axonal injury, whereas ERK activation is downstream of Neu3 sialidase activation. I, A–E, Fluorescent photomicrographs of adult DRG axons uncut (A, C) and 15 min after axotomy (B, D), double-labeled for P38MAPK (red) and phospho P38MAPK (green) under normal conditions (control; A, B) and after removal of eCa2+ (without Ca2+; C, D). The ratio of phospho P38MAPK/P38MAPK intensities (± SEM) normalized against background intensity levels is plotted in E (30–40 axons/chamber, 2 DRGs/chamber, n = 4–6 chambers). II, Activation of P38MAPK using anisomycin increases Neu3 sialidase activity even in the absence of eCa2+. F–H, Fluorescent photomicrographs of uncut adult DRG axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red) and under normal conditions (control; F), after removal of eCa2+ (without Ca2+; G) and after exposure to anisomycin (AIS) in the absence of eCa2+ (without Ca2+ + AIS; H). The ratio of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels is plotted in I (30–40 axons/chamber, 2 DRGs/chamber, n = 4 chambers). III, ERK activation is downstream of Neu3 sialidase activation. J–L, Fluorescent photomicrographs of adult uncut DRG axons, labeled for pERK under control conditions (J) and after exposure to exogenous sialidase (K). The ratio of pERK/ERK intensities (± SEM) normalized against background intensity levels is plotted in L (30–40 axons/chamber, containing 2 DRGs, n = 6 chambers). ***p < 0.001 (Student's t test, two-tailed). **p < 0.01 (Student's t test, two-tailed). Scale bars, 50 μm.

    Techniques Used: Activation Assay, Labeling, Activity Assay, Two Tailed Test

    P38MAPK and ERK are not activated within 15 min after axotomy in retinal axons, but forced activation of P38MAPK increases Neu3 sialidase activity in retinal axons. I, A–D, Fluorescent photomicrographs of adult retinal axons uncut (A,C) and 15 min after axotomy (B,D), double-labeled for P38MAPK (A,B) or ERK (C,D) (red) and pP38MAPK (A,B) or pERK (C,D) (green). Ratios of pP38MAPK/P38MAPK and pERK/ERK intensities (± SEM) normalized against background intensity levels are plotted in E and F (30–40 axons/chamber, n = 4 chambers). II, G, H, Fluorescent photomicrographs of adult uncut retinal axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red). Anisomycin increased the GM1/GD1a ratio (compare H with G). I, Ratios of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels are plotted in I (30–40 axons/chamber, n ≥ 7). ***p < 0.001 (Student's t test, 2-tailed). Scale bars, 50 μm.
    Figure Legend Snippet: P38MAPK and ERK are not activated within 15 min after axotomy in retinal axons, but forced activation of P38MAPK increases Neu3 sialidase activity in retinal axons. I, A–D, Fluorescent photomicrographs of adult retinal axons uncut (A,C) and 15 min after axotomy (B,D), double-labeled for P38MAPK (A,B) or ERK (C,D) (red) and pP38MAPK (A,B) or pERK (C,D) (green). Ratios of pP38MAPK/P38MAPK and pERK/ERK intensities (± SEM) normalized against background intensity levels are plotted in E and F (30–40 axons/chamber, n = 4 chambers). II, G, H, Fluorescent photomicrographs of adult uncut retinal axons, double-labeled for GD1a (green) and GM1 gangliosides (CTB, red). Anisomycin increased the GM1/GD1a ratio (compare H with G). I, Ratios of GM1/GD1a ganglioside intensities (± SEM) normalized against background intensity levels are plotted in I (30–40 axons/chamber, n ≥ 7). ***p < 0.001 (Student's t test, 2-tailed). Scale bars, 50 μm.

    Techniques Used: Activation Assay, Activity Assay, Labeling

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