phospho p38 mapk thr180 tyr182 rabbit mab  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 rabbit mab
    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: <t>p38</t> <t>MAPK</t> inhibitor.
    Phospho P38 Mapk Thr180 Tyr182 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho p38 mapk thr180 tyr182 rabbit mab/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho p38 mapk thr180 tyr182 rabbit mab - by Bioz Stars, 2024-05
    86/100 stars

    Images

    1) Product Images from "p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model"

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.391193

    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.
    Figure Legend Snippet: Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Techniques Used: In Vivo, In Vitro

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Techniques Used: Light Microscopy, Comparison

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Techniques Used: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

    p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Techniques Used: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

    p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Techniques Used: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison

    p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Techniques Used: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

    A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.
    Figure Legend Snippet: A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Techniques Used:

    phospho p38 mapk  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Structured Review

    Cell Signaling Technology Inc phospho p38 mapk
    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. <t>MAPK:</t> Mitogen-activated protein kinase; SB202190: <t>p38</t> <t>MAPK</t> inhibitor.
    Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho p38 mapk/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phospho p38 mapk - by Bioz Stars, 2024-05
    86/100 stars

    Images

    1) Product Images from "p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model"

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.391193

    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.
    Figure Legend Snippet: Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Techniques Used: In Vivo, In Vitro

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Techniques Used: Light Microscopy, Comparison

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Techniques Used: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

    p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Techniques Used: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

    p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Techniques Used: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison

    p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.
    Figure Legend Snippet: p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Techniques Used: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

    A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.
    Figure Legend Snippet: A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Techniques Used:

    rabbit anti phospho p38 mapk  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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  • 86

    Structured Review

    Cell Signaling Technology Inc rabbit anti phospho p38 mapk
    A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 <t>p38</t> <t>MAPK</t> and <t>P38</t> <t>MAPK</t> in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 <t>p38</t> <t>MAPK/P38</t> MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.
    Rabbit Anti Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling"

    Article Title: Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

    Journal: bioRxiv

    doi: 10.1101/2024.05.03.592332

    A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.
    Figure Legend Snippet: A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Confocal Microscopy, Membrane, Western Blot, MANN-WHITNEY

    A , Live cell imaging and model for EGFR (green) translocation from the membrane to the endosomes in HeLa WT (top) and EZR −/− (bottom) cells without EGF stimulation (T0) and with a progressive EGF stimulation (from T10’’ to T60’’). White boxes are magnifications that depict EGFR protein migration. Scale bar 1 µm). Please refer to Video 1. B , IEM (anti-GFP immunolabelling) of cycloheximide treated HeLa WT, WT + EGF, EZR −/− and EZR −/− + EGF cells expressing EGFR-GFP. Endosomes containing EGFR is shown in green. Scale bar 200 nm. Quantitative analysis (right) of EGFR positive endosomes expressed as mean ± SEM. Statistical test: generalized Linear Model with Likelihood Ratio (Poisson Regression). C , immunoblots and calculated levels (bottom) of HER2, pY845 EGFR, EGFR, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells with (+) and without (-) EGF stimulation. Data are expressed as mean of pY845EGFR/EGFR and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: Unpaired t-test for HER2 WT, HER2 EZR −/− , pY845 EGFR EZR −/− , EGFR WT, EGFR EZR −/− , pT180/pY182 p38 MAPK WT, pT180/pY182 p38 MAPK EZR −/− ; unpaired t-test with Welch’s correction for pY845 EGFR WT. D , Representative immunoblots of EGFR in membrane (top) and endosomes (bottom) proteins in HeLa WT and EZR −/− with (+) and without (-) EGF stimulation. ZO-1 and EEA1 are used as membrane and endosomes extraction control, respectively. GAPDH are used as loading control.
    Figure Legend Snippet: A , Live cell imaging and model for EGFR (green) translocation from the membrane to the endosomes in HeLa WT (top) and EZR −/− (bottom) cells without EGF stimulation (T0) and with a progressive EGF stimulation (from T10’’ to T60’’). White boxes are magnifications that depict EGFR protein migration. Scale bar 1 µm). Please refer to Video 1. B , IEM (anti-GFP immunolabelling) of cycloheximide treated HeLa WT, WT + EGF, EZR −/− and EZR −/− + EGF cells expressing EGFR-GFP. Endosomes containing EGFR is shown in green. Scale bar 200 nm. Quantitative analysis (right) of EGFR positive endosomes expressed as mean ± SEM. Statistical test: generalized Linear Model with Likelihood Ratio (Poisson Regression). C , immunoblots and calculated levels (bottom) of HER2, pY845 EGFR, EGFR, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells with (+) and without (-) EGF stimulation. Data are expressed as mean of pY845EGFR/EGFR and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: Unpaired t-test for HER2 WT, HER2 EZR −/− , pY845 EGFR EZR −/− , EGFR WT, EGFR EZR −/− , pT180/pY182 p38 MAPK WT, pT180/pY182 p38 MAPK EZR −/− ; unpaired t-test with Welch’s correction for pY845 EGFR WT. D , Representative immunoblots of EGFR in membrane (top) and endosomes (bottom) proteins in HeLa WT and EZR −/− with (+) and without (-) EGF stimulation. ZO-1 and EEA1 are used as membrane and endosomes extraction control, respectively. GAPDH are used as loading control.

    Techniques Used: Live Cell Imaging, Translocation Assay, Membrane, Migration, Expressing, Western Blot, Extraction

    phospho p38 mapk thr180 tyr182 antibody  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 antibody
    Treatment of cells with Aβ led to increased phospho/total <t>p38</t> <t>MAPK</t> ratio, while blocking <t>p38</t> <t>MAPK</t> activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.
    Phospho P38 Mapk Thr180 Tyr182 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner"

    Article Title: Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms25095033

    Treatment of cells with Aβ led to increased phospho/total p38 MAPK ratio, while blocking p38 MAPK activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.
    Figure Legend Snippet: Treatment of cells with Aβ led to increased phospho/total p38 MAPK ratio, while blocking p38 MAPK activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.

    Techniques Used: Blocking Assay, Activity Assay, Western Blot, Transfection, Software, MANN-WHITNEY, Comparison

    Incubation of cells with ACh reduced the activity of p53 in A549 cells, diminished the activity of p38 MAPK, and enhanced the PKC activity in both A549 and H1299 cells, while co-incubation with Aβ reduced those effects. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight (Control, 0 hour). The cells were then treated as indicated with ACh (100 nM) and in combination with Aβ 1–40/42 or fragments (10 μM). The p53 activity ( A ) in A549 cells and the activity of p38 MAPK ( B , C ) and PKC ( D , E ) were measured as described in the Materials and Methods Section. The data were expressed as the percentage of control by expressing each point relative to the control (set to 100%). The data were then plotted as a function of time using the GraphPad Prism 10.1.1 software. Data were expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.
    Figure Legend Snippet: Incubation of cells with ACh reduced the activity of p53 in A549 cells, diminished the activity of p38 MAPK, and enhanced the PKC activity in both A549 and H1299 cells, while co-incubation with Aβ reduced those effects. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight (Control, 0 hour). The cells were then treated as indicated with ACh (100 nM) and in combination with Aβ 1–40/42 or fragments (10 μM). The p53 activity ( A ) in A549 cells and the activity of p38 MAPK ( B , C ) and PKC ( D , E ) were measured as described in the Materials and Methods Section. The data were expressed as the percentage of control by expressing each point relative to the control (set to 100%). The data were then plotted as a function of time using the GraphPad Prism 10.1.1 software. Data were expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.

    Techniques Used: Incubation, Activity Assay, Expressing, Software

    Representation of the main hypothesis and findings of this study. Aβ decreases the levels of ACh in the media via the activation of p53/AChE and p38 MAPK and/or via blocking the activity of PKC, leading to decreased cell survival. In this model, ACh reverses the effects of Aβ.
    Figure Legend Snippet: Representation of the main hypothesis and findings of this study. Aβ decreases the levels of ACh in the media via the activation of p53/AChE and p38 MAPK and/or via blocking the activity of PKC, leading to decreased cell survival. In this model, ACh reverses the effects of Aβ.

    Techniques Used: Activation Assay, Blocking Assay, Activity Assay

    phospho p38 mapk thr180 tyr182  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182
    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against <t>phospho-p38</t> (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Phospho P38 Mapk Thr180 Tyr182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions"

    Article Title: The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103174

    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against phospho-p38 (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against phospho-p38 (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Activity Assay, Membrane, Western Blot, Expressing, Cell Culture, Staining, Labeling, MANN-WHITNEY

    The activation of Tor1 in cells lacking Rpl2702 depends on Sty1 but not vice versa. (A) Western blotting analysis of Sty1 activity in rpl2702 Δ and rpl2702 Δ tor1 Δ cells expressing Sty1-HA. Antibodies against phospho-p38 and HA were used. Seven sets of experiments were performed. The intensity ratio of phospho-p38 over HA in (A) was quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) In vitro kinase assays of Tor1 activity in rpl2702Δ and rpl2702 Δ sty1 Δ cells. A representative result from four independent experiments was shown here. Note that the absence of Rpl2702, but not the absence of both Rpl2702 and Sty1, increased mTORC2 activity in the reaction containing ATP. (C) Western blotting analysis of mTORC1/Tor2 activity in Psk1-GFP-expressing WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δcells. Antibodies against phospho-p70 S6K (phospho-Psk1-GFP in the graph) and GFP were used. Note that no mTORC1 activity was detected in cells lacking sty1 (MAPK). A representative result from three independent experiments were shown here. The intensity ratio of phospho-p70 S6K over GFP is quantified on the right. The p value was calculated using One-way Anova with the Tukey HSD Post Hoc test, and bars indicate the mean (D) Diagram showing the interplay between Tor1 (i.e., mTORC2). and Sty1 (i.e., MAPK) in cells lacking Rpl2702. The absence of Rpl2702 activates Tor1, and Sty1, and Tor1 activation depends on Sty1 but not vice versa.
    Figure Legend Snippet: The activation of Tor1 in cells lacking Rpl2702 depends on Sty1 but not vice versa. (A) Western blotting analysis of Sty1 activity in rpl2702 Δ and rpl2702 Δ tor1 Δ cells expressing Sty1-HA. Antibodies against phospho-p38 and HA were used. Seven sets of experiments were performed. The intensity ratio of phospho-p38 over HA in (A) was quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) In vitro kinase assays of Tor1 activity in rpl2702Δ and rpl2702 Δ sty1 Δ cells. A representative result from four independent experiments was shown here. Note that the absence of Rpl2702, but not the absence of both Rpl2702 and Sty1, increased mTORC2 activity in the reaction containing ATP. (C) Western blotting analysis of mTORC1/Tor2 activity in Psk1-GFP-expressing WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δcells. Antibodies against phospho-p70 S6K (phospho-Psk1-GFP in the graph) and GFP were used. Note that no mTORC1 activity was detected in cells lacking sty1 (MAPK). A representative result from three independent experiments were shown here. The intensity ratio of phospho-p70 S6K over GFP is quantified on the right. The p value was calculated using One-way Anova with the Tukey HSD Post Hoc test, and bars indicate the mean (D) Diagram showing the interplay between Tor1 (i.e., mTORC2). and Sty1 (i.e., MAPK) in cells lacking Rpl2702. The absence of Rpl2702 activates Tor1, and Sty1, and Tor1 activation depends on Sty1 but not vice versa.

    Techniques Used: Activation Assay, Western Blot, Activity Assay, Expressing, In Vitro

    phospho p38 mapk thr180 tyr182  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk
    A-D . CAL-1 and stably transduced (ShCTRL, shSLAMF7, ShSLAMF8) CAL-1 cells were infected with DsRed WT S. Typhimurium (M.O.I. of 25) for 3 h. Then, cells were fixed, permeabilized, and stained for Phospho-flow cytometry or confocal microscopy. A. Heatmap showing MFI levels for P-IRF3, P-NF- κB p65, P-IRF7, P-ERK-1/2, <t>P-p38,</t> P-STAT1 and P-AKT of Salmonella -infected cells at 3 h p.i. relative to Mock condition. Multiple comparison Kruskal-Wallis test, followed by post-hoc Dunn’s test. n=4. B. Column graphs showing relative MFI of phosphorylated proteins (left, P-NF-κB p65; middle, P-STAT-1; right, P-IRF7) in infected cells. Mean ± SD. n=5. C. Left, representative confocal images of P-NF-κB p65 in CAL-1 and stably transduced CAL-1 cells infected with Salmonella . Scale bars: 20 μm. Right, quantification of P-NF-κB p65 + cells. n=3. One-way ANOVA followed by Dunnett’s multiple comparison test. D. Left, representative flow cytometry histogram of EAT-2 adaptor expression (fluorescence intensity (FI) in CAL-1 cells with positivity indicated by a vertical grey bar. Right, percentages of EAT- 2 + CAL-1 and stably transduced cells infected (Green) or not (Grey) with S. Typhimurium. n=3. E. CAL- 1 and stably transduced CAL-1 cells were infected with DsRed WT Salmonella Typhimurium (M.O.I. of 25) for 1 h. After washes and resuspension in gentamicin-containing medium, cells were treated with MitoTEMPO (100µM) for 2 h. Phospho-flow cytometry was performed to analyze the phosphorylation levels of selected proteins. Column graphs show relative MFI of P-NF-κB p65 (left), P-STAT-1 (middle) and P-IRF7 (right). Mean ± SD. n=4. Two-way ANOVA followed by Sidak’s multiple comparison test. Only statistical differences are shown. *, p < 0.05; **, p < 0.01; *** p < 0.001; **** p < 0.0001. no p value, non-significant. F. Proposed model for SLAMF7 and SLAMF8 function in human pDC during intracellular bacterial infection. At the resting state, few SLAMF8 is expressed on the surface of human pDC in contrast to higher SLAMF7 levels. Infection with an intracellular bacterium, like Salmonella , engages SLAMF7 and SLAMF8 receptors, and elicits the phosphorylation and activation of IRF7, STAT- 1 and NF-κB in a SLAMF7/8-dependent manner . This induces the expression of SLAMF7, SLAMF8, pDC activation markers, as well as cytokine secretion (TNF-α, IL-6 and type I and III IFN) . Elevated levels of SLAMF7 or SLAMF8 restrain mitochondrial ROS production, and subsequently favor NF-κB activation as well as Salmonella persistence . This illustration was created using Biorender.
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    1) Product Images from "SLAMF7 and SLAMF8 receptors shape human plasmacytoid dendritic cell responses to intracellular bacteria"

    Article Title: SLAMF7 and SLAMF8 receptors shape human plasmacytoid dendritic cell responses to intracellular bacteria

    Journal: bioRxiv

    doi: 10.1101/2024.04.25.591064

    A-D . CAL-1 and stably transduced (ShCTRL, shSLAMF7, ShSLAMF8) CAL-1 cells were infected with DsRed WT S. Typhimurium (M.O.I. of 25) for 3 h. Then, cells were fixed, permeabilized, and stained for Phospho-flow cytometry or confocal microscopy. A. Heatmap showing MFI levels for P-IRF3, P-NF- κB p65, P-IRF7, P-ERK-1/2, P-p38, P-STAT1 and P-AKT of Salmonella -infected cells at 3 h p.i. relative to Mock condition. Multiple comparison Kruskal-Wallis test, followed by post-hoc Dunn’s test. n=4. B. Column graphs showing relative MFI of phosphorylated proteins (left, P-NF-κB p65; middle, P-STAT-1; right, P-IRF7) in infected cells. Mean ± SD. n=5. C. Left, representative confocal images of P-NF-κB p65 in CAL-1 and stably transduced CAL-1 cells infected with Salmonella . Scale bars: 20 μm. Right, quantification of P-NF-κB p65 + cells. n=3. One-way ANOVA followed by Dunnett’s multiple comparison test. D. Left, representative flow cytometry histogram of EAT-2 adaptor expression (fluorescence intensity (FI) in CAL-1 cells with positivity indicated by a vertical grey bar. Right, percentages of EAT- 2 + CAL-1 and stably transduced cells infected (Green) or not (Grey) with S. Typhimurium. n=3. E. CAL- 1 and stably transduced CAL-1 cells were infected with DsRed WT Salmonella Typhimurium (M.O.I. of 25) for 1 h. After washes and resuspension in gentamicin-containing medium, cells were treated with MitoTEMPO (100µM) for 2 h. Phospho-flow cytometry was performed to analyze the phosphorylation levels of selected proteins. Column graphs show relative MFI of P-NF-κB p65 (left), P-STAT-1 (middle) and P-IRF7 (right). Mean ± SD. n=4. Two-way ANOVA followed by Sidak’s multiple comparison test. Only statistical differences are shown. *, p < 0.05; **, p < 0.01; *** p < 0.001; **** p < 0.0001. no p value, non-significant. F. Proposed model for SLAMF7 and SLAMF8 function in human pDC during intracellular bacterial infection. At the resting state, few SLAMF8 is expressed on the surface of human pDC in contrast to higher SLAMF7 levels. Infection with an intracellular bacterium, like Salmonella , engages SLAMF7 and SLAMF8 receptors, and elicits the phosphorylation and activation of IRF7, STAT- 1 and NF-κB in a SLAMF7/8-dependent manner . This induces the expression of SLAMF7, SLAMF8, pDC activation markers, as well as cytokine secretion (TNF-α, IL-6 and type I and III IFN) . Elevated levels of SLAMF7 or SLAMF8 restrain mitochondrial ROS production, and subsequently favor NF-κB activation as well as Salmonella persistence . This illustration was created using Biorender.
    Figure Legend Snippet: A-D . CAL-1 and stably transduced (ShCTRL, shSLAMF7, ShSLAMF8) CAL-1 cells were infected with DsRed WT S. Typhimurium (M.O.I. of 25) for 3 h. Then, cells were fixed, permeabilized, and stained for Phospho-flow cytometry or confocal microscopy. A. Heatmap showing MFI levels for P-IRF3, P-NF- κB p65, P-IRF7, P-ERK-1/2, P-p38, P-STAT1 and P-AKT of Salmonella -infected cells at 3 h p.i. relative to Mock condition. Multiple comparison Kruskal-Wallis test, followed by post-hoc Dunn’s test. n=4. B. Column graphs showing relative MFI of phosphorylated proteins (left, P-NF-κB p65; middle, P-STAT-1; right, P-IRF7) in infected cells. Mean ± SD. n=5. C. Left, representative confocal images of P-NF-κB p65 in CAL-1 and stably transduced CAL-1 cells infected with Salmonella . Scale bars: 20 μm. Right, quantification of P-NF-κB p65 + cells. n=3. One-way ANOVA followed by Dunnett’s multiple comparison test. D. Left, representative flow cytometry histogram of EAT-2 adaptor expression (fluorescence intensity (FI) in CAL-1 cells with positivity indicated by a vertical grey bar. Right, percentages of EAT- 2 + CAL-1 and stably transduced cells infected (Green) or not (Grey) with S. Typhimurium. n=3. E. CAL- 1 and stably transduced CAL-1 cells were infected with DsRed WT Salmonella Typhimurium (M.O.I. of 25) for 1 h. After washes and resuspension in gentamicin-containing medium, cells were treated with MitoTEMPO (100µM) for 2 h. Phospho-flow cytometry was performed to analyze the phosphorylation levels of selected proteins. Column graphs show relative MFI of P-NF-κB p65 (left), P-STAT-1 (middle) and P-IRF7 (right). Mean ± SD. n=4. Two-way ANOVA followed by Sidak’s multiple comparison test. Only statistical differences are shown. *, p < 0.05; **, p < 0.01; *** p < 0.001; **** p < 0.0001. no p value, non-significant. F. Proposed model for SLAMF7 and SLAMF8 function in human pDC during intracellular bacterial infection. At the resting state, few SLAMF8 is expressed on the surface of human pDC in contrast to higher SLAMF7 levels. Infection with an intracellular bacterium, like Salmonella , engages SLAMF7 and SLAMF8 receptors, and elicits the phosphorylation and activation of IRF7, STAT- 1 and NF-κB in a SLAMF7/8-dependent manner . This induces the expression of SLAMF7, SLAMF8, pDC activation markers, as well as cytokine secretion (TNF-α, IL-6 and type I and III IFN) . Elevated levels of SLAMF7 or SLAMF8 restrain mitochondrial ROS production, and subsequently favor NF-κB activation as well as Salmonella persistence . This illustration was created using Biorender.

    Techniques Used: Stable Transfection, Infection, Staining, Flow Cytometry, Confocal Microscopy, Comparison, Expressing, Fluorescence, Activation Assay

    anti phospho p38 mapk 4511  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 rabbit mab
    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: <t>p38</t> <t>MAPK</t> inhibitor.
    Phospho P38 Mapk Thr180 Tyr182 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk
    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. <t>MAPK:</t> Mitogen-activated protein kinase; SB202190: <t>p38</t> <t>MAPK</t> inhibitor.
    Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho p38 mapk
    A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 <t>p38</t> <t>MAPK</t> and <t>P38</t> <t>MAPK</t> in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 <t>p38</t> <t>MAPK/P38</t> MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.
    Rabbit Anti Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 antibody
    Treatment of cells with Aβ led to increased phospho/total <t>p38</t> <t>MAPK</t> ratio, while blocking <t>p38</t> <t>MAPK</t> activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.
    Phospho P38 Mapk Thr180 Tyr182 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182
    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against <t>phospho-p38</t> (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Phospho P38 Mapk Thr180 Tyr182, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho p38 mapk 4511
    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against <t>phospho-p38</t> (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Anti Phospho P38 Mapk 4511, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Cell Signaling Technology Inc anti phospho p38 mapk
    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against <t>phospho-p38</t> (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Anti Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: In Vivo, In Vitro

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: Light Microscopy, Comparison

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

    p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

    p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison

    p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

    A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Article Snippet: The following primary antibodies were used for staining: p38 MAPK rabbit polyclonal antibody (1:200, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), FTL rabbit polyclonal antibody (1:200, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673) and SAT1 rabbit polyclonal antibody (1:200, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK Thr180/Tyr182 rabbit mAb (1:200, Cell Signaling Technology, Cat# 4511); rabbit recombinant anti-GPx4 antibody (1:200, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 rabbit polyclonal antibody (1:200, Thermo Fisher Scientific, Cat# PA1-16893).

    Techniques:

    Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: Schematic of the experimental timelines of in vivo (top) and in vitro (bottom) modeling. MAPK: Mitogen-activated protein kinase; SB202190: p38 MAPK inhibitor.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: In Vivo, In Vitro

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced cell death. (A) The effect of different concentrations of glutamate on R28 cell viability over 24 hours ( n = 5). (B) Light microscopy was used to examine the effect of SB202190 on 10 mM glutamate-treated R28 cells. (C) Effect of SB202190 on the cell viability of R28 cells treated with 10 mM glutamate for 24 hours ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . control group (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. (D) The effect of SB202190 on the morphology of R28 cells was observed under a light microscope. Arrows indicate dead cells. Scale bars: 20 µm.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: Light Microscopy, Comparison

    p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 protects R28 cells from glutamate-induced ferroptosis. (A) Propidium iodide (PI; red) and Hoechst 33342 (blue) staining of R28 cells treated with glutamate, SB202190 and ferrostatin-1 as indicated for 24 hours. Scale bar: 100 µm. (B) Quantification of the results shown in A ( n = 5). (C) Transmission electron microscope images of R28 cells treated with glutamate and SB202190. Scale bar: 1 µm. (D) R28 cells treated as indicated were stained with the fluorescent probe Mito -FerroOrange to determine ferrous ion levels. FerroOrange undergoes an irreversible reaction with intracellular Fe 2+ in live cells, resulting in orange fluorescence emission. Ex: 543 nm and Em: 580 nm. Scale bar: 50 µm. (E) Quantification of mean density of FerroOrange fluorescence in the groups ( n = 5). (F) R28 cells treated as indicated were stained with Liperfluo for measurement of lipid peroxides. Liperfluo is selectively oxidized by lipid peroxides, and the oxidized form of Liperfluo exhibits strong green fluorescence at the cell membrane. Scale bar: 50 µm. (G) Comparison of mean density of Liperfluor fluorescence in the groups ( n = 5). (H, I) Malondialdehyde (MDA; H) and glutathione (GSH; I) levels in R28 cells after glutamate and SB202190 treatment ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: Staining, Transmission Assay, Microscopy, Fluorescence, Membrane, Comparison

    p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 regulates ferroptosis in a glutamate R28 cell model by regulating FTL and SAT1. (A) p38 MAPK, p-p38 MAPK, FTL and SAT1 protein expression in R28 cells treated with glutamate and SB202190 for 24 hours ( n = 3). (B–D) Quantification analysis of p-p38 MAPK/ p38 MAPK, FTL and SAT1 protein expression in R28 cells (n = 3). (E–I) Three days after intravitreal injection of NMDA and SB202190, paraffin sections were collected and processed for immunofluorescence experiments to measure the fluorescence intensity of p-p38 MAPK (E, F), FTL (G, H), and SAT1 (I, J) of the retinal sections in each group under a fluorescence microscope ( n = 3). Scale bars: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were repeated. FTL: Ferritin light chain; MAPK: mitogen activated protein kinases.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: Expressing, Injection, Immunofluorescence, Fluorescence, Microscopy, Comparison

    p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 alleviates lipid peroxidation by regulating the SLC7A11/GSH/GPx4 pathway. (A, B) Flow cytometry was used to detect the effect of SB202190 on lipid reactive oxygen species production in R28 cells treated as indicated for 24 hours ( n = 4). (C–E) SLC7A11 and GPx4 protein expressions in R28 cells were detected by western blotting ( n = 3). (F–I) Fluorescence microscopy was performed to evaluate the fluorescence intensity changes of and GPx4 (F, G) and SLC7A11 (H, I) in rat retinal sections after intraluminal injection of NMDA and SB202190 for 3 days ( n = 3). Scale bars in F and H: 10 µm. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. GPx4: Glutathione peroxidase 4; GSH: glutathione; MAPK: mitogen-activated protein kinase.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: Flow Cytometry, Western Blot, Fluorescence, Microscopy, Injection, Comparison

    p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: p38 MAPK inhibitor SB202190 attenuates N-methyl-D-aspartate (NMDA)-induced damage in the rat retina. (A, B) Qualitative observation (A) and quantitative analysis (B) of the effects of SB202190 on NMDA-induced retinal ganglion cell (RGC) injury in rat retina ( n = 5). Three days after intravitreal injection of NMDA and SB202190, retinal plating was performed. RGCs were fluorescently labeled with Brn3a antibody and surviving RGC exhibited strong green fluorescence under a fluorescence microscope. Scale bar: 50 μm. (C) Effects of SB202190 on retinal morphology in NMDA model rats. Hematoxylin and eosin staining was performed 3 days after intravitreal injection of NMDA and SB202190. Scale bar: 50 µm. (D) Effect of SB202190 on RGC density (cells/100 µm) in NMDA model rats 3 days after intravitreal injection ( n = 4). (E, F) Thickness of the retinal ganglion cell body complex (GCC) at 500, 1000, 1500, and 2000 µm from the optic disc 3 days after intravitreal injection of NMDA and SB202190. (G–I) Flash visual evoked potentials of rats 3 days after intravitreal injection of NMDA and SB202190 ( n = 4). GCL: Ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; ONL: outer nuclear layer; RGC: retinal ganglion cell. Inner retina consists of GCL and IPL. ** P < 0.01, **** P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple comparison test). Data are expressed as the means ± SD. At least three independent experiments were performed. MAPK: Mitogen-activated protein kinase.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques: Injection, Labeling, Fluorescence, Microscopy, Staining, Comparison

    A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Journal: Neural Regeneration Research

    Article Title: p38 MAPK inhibitor SB202190 suppresses ferroptosis in the glutamate-induced retinal excitotoxicity glaucoma model

    doi: 10.4103/1673-5374.391193

    Figure Lengend Snippet: A schematic model for the mechanism of p38 MAPK inhibitor SB202190 in alleviating ferroptosis in the glutamate-induced retinal excitotoxic glaucoma model. The p38 MAPK inhibitor sB202190 inhibits ferroptosis in the glutamatergic excitotoxicity glaucoma model by upregulation of ferritin light chain (FTL), downregulation of SAT1 and regulation of the SLC7A11/GSH/GPx4 signaling pathway. GPx4: Glutathione peroxidase 4; GSH: glutathione; GSSG: glutathione disulfide; MAPK: mitogen-activated protein kinase; ROS: reactive oxygen species.

    Article Snippet: The membrane were incubated with blocking buffer (5% nonfat milk in TBST) for 90 minutes at room temperature, followed by incubation with primary antibodies overnight at 4°C: beta-actin monoclonal antibody (1:1000, Proteintech, Rosemont, IL, USA, Cat# 66009-1-Ig, RRID: AB_2687938), p38 MAPK polyclonal antibody (1:1000, Proteintech, Cat# 14064-1-AP, RRID: AB_2878007), ferritin light chain (FTL) polyclonal antibody (1:1000, Proteintech, Cat# 10727-1-AP, RRID: AB_2278673), and SAT1 polyclonal antibody (1:1000, Proteintech, Cat# 10708-1-AP, RRID: AB_2877739); phospho-p38 MAPK (1:1000, Thr180/Tyr182 Rabbit mAb, Cell Signaling Technology, Danvers, MA, USA, Cat# 4511); recombinant anti-glutathione peroxidase 4 (GPx4) antibody (1:1000, Abcam, Cat# ab125066, RRID: AB_10973901); and SLC7A11 polyclonal antibody (1:1000, Thermo Fisher Scientific, PA1-16893).

    Techniques:

    A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.

    Journal: bioRxiv

    Article Title: Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

    doi: 10.1101/2024.05.03.592332

    Figure Lengend Snippet: A, Bubble plot representing the enrichment analysis of 530 DEGs performed in SILAC Phosphoproteomics data. Color and x axis represent minus logarithms of p Value. The size represents numbers of genes enriched in the indicated data. B , Physical interactions, obtained by GeneMANIA, highlight Ezrin and EGFR binding. C , Volcano plot of DEGs, with up-regulated EGFR and down-regulated MAP2 and ERBB2 (no threshold on Log2FC and 0.05 threshold on -Log10FDR). Legend: red dot, up-regulated gene; blue dot, down-regulated gene; grey dot, not significant gene. D, Co-IP data for Ezrin-EGFR interaction. For the co-IP analyses, was used Ezrin antibody, conjugated with beads, and immunoblotted with EGFR antibody for WT and EZR −/− (left) and HeLa EZR T567D and EZR T567A (right) HeLa cells, respectively. Schematic representation of HeLa EZR T567D and EZR T567A co-IP (bottom). E , Confocal microscopy images showing EGFR (green) and EZR (red) co-localization on the membrane in HeLa WT cells (left) and magnified views of the regions are provided (right). Scale bar 10 µm (magnification 1 µm). Representative plots of co-localization profiles on the membrane between EGFR (green) and EZR (red). F , Immunofluorescent labelling images of EGFR in HeLa WT and EZR −/− cells, observed by confocal microscopy. Scale bar 10 µm. G , Immunoblots and calculated levels (bottom) of HER3, pY845 EGFR, EGFR, pT222 MK2, MK2, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells. Data are expressed as mean of pY845EGFR/EGFR, pT222 MK2/MK2 and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: unpaired t-test for pY845 EGFR; Mann-Whitney test for HER3, EGFR, pT222 MK2, pT180/pY182 p38 MAPK.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti-NBR1 (1:1000, Abnova MO1), rabbit anti-LAMP1 (1:500, Sigma L1418), mouse anti-Ezrin (1:1000, Novex 357300), mouse anti-SQSTM1/P62 (1:1000, Abcam ab56416), rabbit anti-Cathepsin D (1:1000, Cell Signaling 2284), rabbit anti-LC3 (1:1000, Novus NB100-2220), mouse anti-GAPDH (1:1000, Santa Cruz SC-32233), rabbit anti-HER2/ErbB2 (1:1000, Cell Signaling 2165), rabbit anti-HER3/ErbB3 (1:1000, Cell Signaling 12708), rabbit anti-phospho-EGF receptor (Tyr845) (1:1000, Cell Signaling 6963), rabbit anti-EGF receptor (1:1000, Cell Signaling 4267), rabbit anti-MAPKAPK-2 (1:1000, Cell Signaling 3042), rabbit anti-phospho-MAPKAPK-2 (Thr222) (1:1000, Cell Signaling 3316), rabbit anti-p38 MAPK (1:1000, Cell Signaling 8690), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling 4511), rabbit anti-ZO1 (1:1000, Abcam ab216880), mouse anti-EEA1 (1:1000, BD 610457), rabbit anti-Tuberin/TSC2 (1:1000, Cell Signaling 4308), rabbit anti-phospho-Tuberin/TSC2 (Ser939) (1:1000, Cell Signaling 3615), rabbit anti-phospho-Tuberin/TSC2 (Thr1462) (1:1000, Cell Signaling 3617), rabbit anti-p70 S6 Kinase (1:1000, Cell Signaling 9202), mouse anti-phospho-p70 S6 Kinase (Thr389) (1:1000, Cell Signaling 9206), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-4E-BP1 (1:1000, Cell Signaling 9644), rabbit anti-phospho-4E-BP1 (Ser65) (1:1000, Cell Signaling 9456), rabbit anti-phospho-4E-BP1 (Thr37/46) (1:1000, Cell Signaling 2855), rabbit anti-Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti-EGFR (1:500, Santa Cruz sc-120), mouse anti-p-EGFR (1:500, Santa Cruz sc-57542).

    Techniques: Binding Assay, Co-Immunoprecipitation Assay, Confocal Microscopy, Membrane, Western Blot, MANN-WHITNEY

    A , Live cell imaging and model for EGFR (green) translocation from the membrane to the endosomes in HeLa WT (top) and EZR −/− (bottom) cells without EGF stimulation (T0) and with a progressive EGF stimulation (from T10’’ to T60’’). White boxes are magnifications that depict EGFR protein migration. Scale bar 1 µm). Please refer to Video 1. B , IEM (anti-GFP immunolabelling) of cycloheximide treated HeLa WT, WT + EGF, EZR −/− and EZR −/− + EGF cells expressing EGFR-GFP. Endosomes containing EGFR is shown in green. Scale bar 200 nm. Quantitative analysis (right) of EGFR positive endosomes expressed as mean ± SEM. Statistical test: generalized Linear Model with Likelihood Ratio (Poisson Regression). C , immunoblots and calculated levels (bottom) of HER2, pY845 EGFR, EGFR, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells with (+) and without (-) EGF stimulation. Data are expressed as mean of pY845EGFR/EGFR and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: Unpaired t-test for HER2 WT, HER2 EZR −/− , pY845 EGFR EZR −/− , EGFR WT, EGFR EZR −/− , pT180/pY182 p38 MAPK WT, pT180/pY182 p38 MAPK EZR −/− ; unpaired t-test with Welch’s correction for pY845 EGFR WT. D , Representative immunoblots of EGFR in membrane (top) and endosomes (bottom) proteins in HeLa WT and EZR −/− with (+) and without (-) EGF stimulation. ZO-1 and EEA1 are used as membrane and endosomes extraction control, respectively. GAPDH are used as loading control.

    Journal: bioRxiv

    Article Title: Ezrin defines TSC1 activation at endosomal compartments through EGFR-AKT signaling

    doi: 10.1101/2024.05.03.592332

    Figure Lengend Snippet: A , Live cell imaging and model for EGFR (green) translocation from the membrane to the endosomes in HeLa WT (top) and EZR −/− (bottom) cells without EGF stimulation (T0) and with a progressive EGF stimulation (from T10’’ to T60’’). White boxes are magnifications that depict EGFR protein migration. Scale bar 1 µm). Please refer to Video 1. B , IEM (anti-GFP immunolabelling) of cycloheximide treated HeLa WT, WT + EGF, EZR −/− and EZR −/− + EGF cells expressing EGFR-GFP. Endosomes containing EGFR is shown in green. Scale bar 200 nm. Quantitative analysis (right) of EGFR positive endosomes expressed as mean ± SEM. Statistical test: generalized Linear Model with Likelihood Ratio (Poisson Regression). C , immunoblots and calculated levels (bottom) of HER2, pY845 EGFR, EGFR, pT180/pY182 p38 MAPK and P38 MAPK in HeLa WT and EZR −/− cells with (+) and without (-) EGF stimulation. Data are expressed as mean of pY845EGFR/EGFR and pT180/pY182 p38 MAPK/P38 MAPK ratio ± SEM (n=3 experiments at least). GAPDH was used as loading control. Statistical test: Unpaired t-test for HER2 WT, HER2 EZR −/− , pY845 EGFR EZR −/− , EGFR WT, EGFR EZR −/− , pT180/pY182 p38 MAPK WT, pT180/pY182 p38 MAPK EZR −/− ; unpaired t-test with Welch’s correction for pY845 EGFR WT. D , Representative immunoblots of EGFR in membrane (top) and endosomes (bottom) proteins in HeLa WT and EZR −/− with (+) and without (-) EGF stimulation. ZO-1 and EEA1 are used as membrane and endosomes extraction control, respectively. GAPDH are used as loading control.

    Article Snippet: For Western blot analysis, the following antibodies were used: mouse anti-NBR1 (1:1000, Abnova MO1), rabbit anti-LAMP1 (1:500, Sigma L1418), mouse anti-Ezrin (1:1000, Novex 357300), mouse anti-SQSTM1/P62 (1:1000, Abcam ab56416), rabbit anti-Cathepsin D (1:1000, Cell Signaling 2284), rabbit anti-LC3 (1:1000, Novus NB100-2220), mouse anti-GAPDH (1:1000, Santa Cruz SC-32233), rabbit anti-HER2/ErbB2 (1:1000, Cell Signaling 2165), rabbit anti-HER3/ErbB3 (1:1000, Cell Signaling 12708), rabbit anti-phospho-EGF receptor (Tyr845) (1:1000, Cell Signaling 6963), rabbit anti-EGF receptor (1:1000, Cell Signaling 4267), rabbit anti-MAPKAPK-2 (1:1000, Cell Signaling 3042), rabbit anti-phospho-MAPKAPK-2 (Thr222) (1:1000, Cell Signaling 3316), rabbit anti-p38 MAPK (1:1000, Cell Signaling 8690), rabbit anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000, Cell Signaling 4511), rabbit anti-ZO1 (1:1000, Abcam ab216880), mouse anti-EEA1 (1:1000, BD 610457), rabbit anti-Tuberin/TSC2 (1:1000, Cell Signaling 4308), rabbit anti-phospho-Tuberin/TSC2 (Ser939) (1:1000, Cell Signaling 3615), rabbit anti-phospho-Tuberin/TSC2 (Thr1462) (1:1000, Cell Signaling 3617), rabbit anti-p70 S6 Kinase (1:1000, Cell Signaling 9202), mouse anti-phospho-p70 S6 Kinase (Thr389) (1:1000, Cell Signaling 9206), rabbit anti-Akt (1:1000, Cell Signaling 9272), rabbit anti-phospho-Akt (Ser473) (1:1000, Cell Signaling 4060), rabbit anti-4E-BP1 (1:1000, Cell Signaling 9644), rabbit anti-phospho-4E-BP1 (Ser65) (1:1000, Cell Signaling 9456), rabbit anti-phospho-4E-BP1 (Thr37/46) (1:1000, Cell Signaling 2855), rabbit anti-Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti-EGFR (1:500, Santa Cruz sc-120), mouse anti-p-EGFR (1:500, Santa Cruz sc-57542).

    Techniques: Live Cell Imaging, Translocation Assay, Membrane, Migration, Expressing, Western Blot, Extraction

    Treatment of cells with Aβ led to increased phospho/total p38 MAPK ratio, while blocking p38 MAPK activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.

    Journal: International Journal of Molecular Sciences

    Article Title: Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner

    doi: 10.3390/ijms25095033

    Figure Lengend Snippet: Treatment of cells with Aβ led to increased phospho/total p38 MAPK ratio, while blocking p38 MAPK activity with SB203580 decreased p53 activity in A549 cells and increased ACh levels in the media of both cell lines. Cells (0.2 × 10) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight. The cells were then treated as indicated for 72 h with Aβ 1–40/42 or fragments (10 μM) ± SB203580 (SB, 20 μM), and then, the p38 MAPK assay ( A ) and Western blotting ( B ) were carried out (Materials and Methods). The p53 activity was measured in A549 cells ( C ). Cells transfected with either control or p53 siRNA were treated for 72 h with Aβ 1–40/42 or fragments ± SB203580; then, the levels of ACh ( D – F ) were measured as described in the Materials and Methods Section. Data from five independent assays, each carried out in triplicate, were averaged, normalized, and expressed as fold change relative to the control of each cell line ( A ) or to control in the absence of SB203580 ( C – F ) using the GraphPad 10.1.1 software. The graphs summarize the results expressed as means ± SD (n = 5). Asterisks indicate a statistically significant difference from control. Absence of asterisks indicates no significance, Mann–Whitney test. Statistical differences between different groups were analyzed by an ordinary one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test, * p < 0.05, ** p < 0.01.

    Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence control siRNA (Unconjugated, 6568), rabbit p53 antibody (9282), p38 MAPK antibody (9212) that detects endogenous levels of total p38α/β/γ MAPK, phospho-p38 MAPK (Thr180/Tyr182) antibody (9211), PKCα antibody (2056), and SB203580 (5633S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Blocking Assay, Activity Assay, Western Blot, Transfection, Software, MANN-WHITNEY, Comparison

    Incubation of cells with ACh reduced the activity of p53 in A549 cells, diminished the activity of p38 MAPK, and enhanced the PKC activity in both A549 and H1299 cells, while co-incubation with Aβ reduced those effects. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight (Control, 0 hour). The cells were then treated as indicated with ACh (100 nM) and in combination with Aβ 1–40/42 or fragments (10 μM). The p53 activity ( A ) in A549 cells and the activity of p38 MAPK ( B , C ) and PKC ( D , E ) were measured as described in the Materials and Methods Section. The data were expressed as the percentage of control by expressing each point relative to the control (set to 100%). The data were then plotted as a function of time using the GraphPad Prism 10.1.1 software. Data were expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.

    Journal: International Journal of Molecular Sciences

    Article Title: Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner

    doi: 10.3390/ijms25095033

    Figure Lengend Snippet: Incubation of cells with ACh reduced the activity of p53 in A549 cells, diminished the activity of p38 MAPK, and enhanced the PKC activity in both A549 and H1299 cells, while co-incubation with Aβ reduced those effects. Cells (0.2 × 10 5 ) were grown in 10% FBS-supplemented media for 24 h and then serum-starved overnight (Control, 0 hour). The cells were then treated as indicated with ACh (100 nM) and in combination with Aβ 1–40/42 or fragments (10 μM). The p53 activity ( A ) in A549 cells and the activity of p38 MAPK ( B , C ) and PKC ( D , E ) were measured as described in the Materials and Methods Section. The data were expressed as the percentage of control by expressing each point relative to the control (set to 100%). The data were then plotted as a function of time using the GraphPad Prism 10.1.1 software. Data were expressed as the mean ± S.D. of three independent experiments, each carried out in triplicate.

    Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence control siRNA (Unconjugated, 6568), rabbit p53 antibody (9282), p38 MAPK antibody (9212) that detects endogenous levels of total p38α/β/γ MAPK, phospho-p38 MAPK (Thr180/Tyr182) antibody (9211), PKCα antibody (2056), and SB203580 (5633S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Incubation, Activity Assay, Expressing, Software

    Representation of the main hypothesis and findings of this study. Aβ decreases the levels of ACh in the media via the activation of p53/AChE and p38 MAPK and/or via blocking the activity of PKC, leading to decreased cell survival. In this model, ACh reverses the effects of Aβ.

    Journal: International Journal of Molecular Sciences

    Article Title: Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner

    doi: 10.3390/ijms25095033

    Figure Lengend Snippet: Representation of the main hypothesis and findings of this study. Aβ decreases the levels of ACh in the media via the activation of p53/AChE and p38 MAPK and/or via blocking the activity of PKC, leading to decreased cell survival. In this model, ACh reverses the effects of Aβ.

    Article Snippet: SignalSilence p53 siRNA I (6231), SignalSilence control siRNA (Unconjugated, 6568), rabbit p53 antibody (9282), p38 MAPK antibody (9212) that detects endogenous levels of total p38α/β/γ MAPK, phospho-p38 MAPK (Thr180/Tyr182) antibody (9211), PKCα antibody (2056), and SB203580 (5633S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, Blocking Assay, Activity Assay

    The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against phospho-p38 (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Journal: Redox Biology

    Article Title: The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions

    doi: 10.1016/j.redox.2024.103174

    Figure Lengend Snippet: The absence of Rpl2702 activates Sty1 and requires Sty1 activity for maintaining mitochondrial membrane potential and mROS levels. (A) Western blotting assays were performed to measure Sty1 activity in WT and rpl2702Δ cells. Both types of cells expressed Sty1-HA. Antibodies against phospho-p38 (the phosphorylation form of Sty1) and HA were used. Data are from seven sets of experiments, and the intensity ratio of phospho-p38 over HA is quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) Testing the protein levels of Sty1 in WT and rpl2702Δ cells and rpl2702Δ cells ectopically expressing Rpl2702 from its own promoter at the leu1 locus. All cells expressed Sty1-HA. Antibodies against HA and Tubulin were used. Data are from four sets of experiments, and the intensity ratio of HA over Tubulin is quantified on the right. The p values were calculated using One-way ANOVA with the Tukey HSD Post Hoc test, and bars indicate the mean. (C) Maximum projection images of WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δ cells. The cells expressed Sdh2-GFP and were cultured in EMM5S. DIC , differential interference contrast. Scale bar, 10 μm. (D) Mitochondrial morphology quantification for the cells in (C). The categories of mitochondrial morphology are indicated. (E) Maximum projection images of the indicated cells stained with DiOC6. Cells were mixed as in C. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-tdTomato-expressing cells, respectively. The average intensity of DiOC6 staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (F) Maximum projection images of the indicated cells stained with MitoSox Red. Cells were mixed as in D. Dashed lines mark cell contours. In the top panel, orange and blue dashed lines indicate WT cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. In the bottom panel, orange and blue dashed lines indicate rpl2702Δ cells (the absence of fluorescent labeling) and rpl2702Δsty1Δ Sid4-GFP-expressing cells, respectively. The average intensity of MitoSox Red staining was measured and quantified (right); the number of cells observed is indicated, and bars indicate the mean. The p values were calculated using the Wilcoxon-Mann-Whitney Rank Sum Test. Three independent experiments were performed, and shown is a representative experiment. Scale bar, 10 μm (G) A diagram is shown to summarize the data in this figure: the absence of Rpl2702 causes mitochondrial fragmentation and increases mitochondrial membrane potential and mROS in a Sty1-dependent manner. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Article Snippet: Samples were separated by SDS-PAGE and then immunoblotted with antibodies against Tubulin (dilution factor, 1:10,000; 63–160, Bio Academia), GFP (dilution factor, 1:2000; 600-101-215, Rockland), FLAG (dilution factor, 1:1000; M20008S, Abmart), Phospho-AMPKα (Thr172) (dilution factor, 1:1000; 2535, Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) (dilution factor, 1:1000; 4511, Cell Signaling Technology), phospho-p70 S6 Kinase (Thr389) (dilution factor, 1:1000; 9206, Cell Signaling Technology), and Dnm1(dilution factor, 1:1000; Homemade).

    Techniques: Activity Assay, Membrane, Western Blot, Expressing, Cell Culture, Staining, Labeling, MANN-WHITNEY

    The activation of Tor1 in cells lacking Rpl2702 depends on Sty1 but not vice versa. (A) Western blotting analysis of Sty1 activity in rpl2702 Δ and rpl2702 Δ tor1 Δ cells expressing Sty1-HA. Antibodies against phospho-p38 and HA were used. Seven sets of experiments were performed. The intensity ratio of phospho-p38 over HA in (A) was quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) In vitro kinase assays of Tor1 activity in rpl2702Δ and rpl2702 Δ sty1 Δ cells. A representative result from four independent experiments was shown here. Note that the absence of Rpl2702, but not the absence of both Rpl2702 and Sty1, increased mTORC2 activity in the reaction containing ATP. (C) Western blotting analysis of mTORC1/Tor2 activity in Psk1-GFP-expressing WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δcells. Antibodies against phospho-p70 S6K (phospho-Psk1-GFP in the graph) and GFP were used. Note that no mTORC1 activity was detected in cells lacking sty1 (MAPK). A representative result from three independent experiments were shown here. The intensity ratio of phospho-p70 S6K over GFP is quantified on the right. The p value was calculated using One-way Anova with the Tukey HSD Post Hoc test, and bars indicate the mean (D) Diagram showing the interplay between Tor1 (i.e., mTORC2). and Sty1 (i.e., MAPK) in cells lacking Rpl2702. The absence of Rpl2702 activates Tor1, and Sty1, and Tor1 activation depends on Sty1 but not vice versa.

    Journal: Redox Biology

    Article Title: The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions

    doi: 10.1016/j.redox.2024.103174

    Figure Lengend Snippet: The activation of Tor1 in cells lacking Rpl2702 depends on Sty1 but not vice versa. (A) Western blotting analysis of Sty1 activity in rpl2702 Δ and rpl2702 Δ tor1 Δ cells expressing Sty1-HA. Antibodies against phospho-p38 and HA were used. Seven sets of experiments were performed. The intensity ratio of phospho-p38 over HA in (A) was quantified on the right. The p value was calculated using Student's t -test, and bars indicate the mean. (B) In vitro kinase assays of Tor1 activity in rpl2702Δ and rpl2702 Δ sty1 Δ cells. A representative result from four independent experiments was shown here. Note that the absence of Rpl2702, but not the absence of both Rpl2702 and Sty1, increased mTORC2 activity in the reaction containing ATP. (C) Western blotting analysis of mTORC1/Tor2 activity in Psk1-GFP-expressing WT, rpl2702 Δ, sty1 Δ and rpl2702 Δ sty1 Δcells. Antibodies against phospho-p70 S6K (phospho-Psk1-GFP in the graph) and GFP were used. Note that no mTORC1 activity was detected in cells lacking sty1 (MAPK). A representative result from three independent experiments were shown here. The intensity ratio of phospho-p70 S6K over GFP is quantified on the right. The p value was calculated using One-way Anova with the Tukey HSD Post Hoc test, and bars indicate the mean (D) Diagram showing the interplay between Tor1 (i.e., mTORC2). and Sty1 (i.e., MAPK) in cells lacking Rpl2702. The absence of Rpl2702 activates Tor1, and Sty1, and Tor1 activation depends on Sty1 but not vice versa.

    Article Snippet: Samples were separated by SDS-PAGE and then immunoblotted with antibodies against Tubulin (dilution factor, 1:10,000; 63–160, Bio Academia), GFP (dilution factor, 1:2000; 600-101-215, Rockland), FLAG (dilution factor, 1:1000; M20008S, Abmart), Phospho-AMPKα (Thr172) (dilution factor, 1:1000; 2535, Cell Signaling Technology), Phospho-p38 MAPK (Thr180/Tyr182) (dilution factor, 1:1000; 4511, Cell Signaling Technology), phospho-p70 S6 Kinase (Thr389) (dilution factor, 1:1000; 9206, Cell Signaling Technology), and Dnm1(dilution factor, 1:1000; Homemade).

    Techniques: Activation Assay, Western Blot, Activity Assay, Expressing, In Vitro