anisomycin  (Alomone Labs)

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.4432-13.2014

Figure Lengend Snippet: P38MAPK activation is the intermediary step between eCa 2+ 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 eCa 2+ (without Ca 2+ ; 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 eCa 2+ . 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 eCa 2+ (without Ca 2+ ; G ) and after exposure to anisomycin (AIS) in the absence of eCa 2+ (without Ca 2+ + 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

Article Snippet: Anisomycin (10 μ m , Alomone Labs) was used to activate P38MAPK.

Techniques: Activation Assay, Labeling, Activity Assay, CtB Assay

Journal: The Journal of Neuroscience

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

doi: 10.1523/JNEUROSCI.4432-13.2014

Figure Lengend 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

Article Snippet: Anisomycin (10 μ m , Alomone Labs) was used to activate P38MAPK.

Techniques: Activation Assay, Activity Assay, Labeling, CtB Assay

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-53419-4

Figure Lengend 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.

Article Snippet: BAM7 was purchased from Adooq BioScience, anisomycin was purchased from Alomone Labs, TNF-α was purchased from PeproTech, NAC was purchased from Wako, and Q-VD-OPH, DPI, LY294002, and CTZ were purchased from Cayman Chemical.

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-53419-4

Figure Lengend 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

Article Snippet: BAM7 was purchased from Adooq BioScience, anisomycin was purchased from Alomone Labs, TNF-α was purchased from PeproTech, NAC was purchased from Wako, and Q-VD-OPH, DPI, LY294002, and CTZ were purchased from Cayman Chemical.

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-53419-4

Figure Lengend 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

Article Snippet: BAM7 was purchased from Adooq BioScience, anisomycin was purchased from Alomone Labs, TNF-α was purchased from PeproTech, NAC was purchased from Wako, and Q-VD-OPH, DPI, LY294002, and CTZ were purchased from Cayman Chemical.

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

Journal: Scientific Reports

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

doi: 10.1038/s41598-019-53419-4

Figure Lengend 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

Article Snippet: BAM7 was purchased from Adooq BioScience, anisomycin was purchased from Alomone Labs, TNF-α was purchased from PeproTech, NAC was purchased from Wako, and Q-VD-OPH, DPI, LY294002, and CTZ were purchased from Cayman Chemical.

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