phospho p38 mapk thr180 tyr182 rabbit mab  (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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    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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    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
    https://www.bioz.com/result/rabbit anti phospho p38 mapk/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho p38 mapk - by Bioz Stars, 2024-05
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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

    rabbit anti phospho p38 antibody  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc rabbit anti phospho p38 antibody
    Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of <t>p38</t> and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.
    Rabbit Anti Phospho P38 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
    https://www.bioz.com/result/rabbit anti phospho p38 antibody/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho p38 antibody - by Bioz Stars, 2024-05
    86/100 stars

    Images

    1) Product Images from "Casein kinase-2 inhibition promotes retinal ganglion cell survival after acute intraocular pressure elevation"

    Article Title: Casein kinase-2 inhibition promotes retinal ganglion cell survival after acute intraocular pressure elevation

    Journal: Neural Regeneration Research

    doi: 10.4103/1673-5374.385310

    Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of p38 and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.
    Figure Legend Snippet: Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of p38 and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.

    Techniques Used: Western Blot, Expressing, Injection

    rabbit anti phospho p38  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc rabbit anti phospho p38
    Rabbit Anti Phospho P38, 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/rabbit anti phospho p38/product/Cell Signaling Technology Inc
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    rabbit anti phospho p38  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho p38

    Rabbit Anti Phospho P38, 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 "Tgif1-deficiency impairs cytoskeletal architecture in osteoblasts by activating PAK3 signaling"

    Article Title: Tgif1-deficiency impairs cytoskeletal architecture in osteoblasts by activating PAK3 signaling

    Journal: eLife

    doi: 10.7554/eLife.94265


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    rabbit anti phospho p38 mapk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho 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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    rabbit anti phospho p38 mapk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho p38 mapk
    OT suppresses the VCR-induced activation of the ERK1/2 and <t>p38</t> <t>MAPK</t> pathways. (A) Representative image of phosphorylated and total ERK1/2 and p38 protein tested using Western blot. (B–C) Quantification of phosphorylated ERK1/2 (B) and p38 (C) relative expression levels (n = 5). The data are expressed as the means ± SEM, and one-way ANOVA combined with the Newman–Keuls test is used to compare the differences among multiple groups. ** p < 0.01 and *** p < 0.001 versus the NS group; ## p < 0.01 and ### p < 0.001 versus the VCR group.
    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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    1) Product Images from "Protective effect of oxytocin on vincristine-induced gastrointestinal dysmotility in mice"

    Article Title: Protective effect of oxytocin on vincristine-induced gastrointestinal dysmotility in mice

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2024.1270612

    OT suppresses the VCR-induced activation of the ERK1/2 and p38 MAPK pathways. (A) Representative image of phosphorylated and total ERK1/2 and p38 protein tested using Western blot. (B–C) Quantification of phosphorylated ERK1/2 (B) and p38 (C) relative expression levels (n = 5). The data are expressed as the means ± SEM, and one-way ANOVA combined with the Newman–Keuls test is used to compare the differences among multiple groups. ** p < 0.01 and *** p < 0.001 versus the NS group; ## p < 0.01 and ### p < 0.001 versus the VCR group.
    Figure Legend Snippet: OT suppresses the VCR-induced activation of the ERK1/2 and p38 MAPK pathways. (A) Representative image of phosphorylated and total ERK1/2 and p38 protein tested using Western blot. (B–C) Quantification of phosphorylated ERK1/2 (B) and p38 (C) relative expression levels (n = 5). The data are expressed as the means ± SEM, and one-way ANOVA combined with the Newman–Keuls test is used to compare the differences among multiple groups. ** p < 0.01 and *** p < 0.001 versus the NS group; ## p < 0.01 and ### p < 0.001 versus the VCR group.

    Techniques Used: Activation Assay, Western Blot, Expressing

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


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    Cell Signaling Technology Inc rabbit polyclonal anti phospho p38 mapk thr180 tyr182 antibody
    Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, <t>phospho-p38,</t> and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.
    Rabbit Polyclonal Anti 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 "Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells"

    Article Title: Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells

    Journal: iScience

    doi: 10.1016/j.isci.2024.109675

    Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.
    Figure Legend Snippet: Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.

    Techniques Used: Cell Culture, Western Blot, Expressing


    Figure Legend Snippet:

    Techniques Used: Recombinant, CCK-8 Assay, Staining, Protease Inhibitor, Saline, Blocking Assay, Ab Array, Software

    rabbit anti phospho p38 mapk t180 y182  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho p38 mapk t180 y182
    Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the <t>p38</t> pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).
    Rabbit Anti Phospho P38 Mapk T180 Y182, 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 "Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling"

    Article Title: Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-58619-1

    Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).
    Figure Legend Snippet: Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).

    Techniques Used: Transformation Assay, Incubation, Sequencing, Western Blot

    MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.
    Figure Legend Snippet: MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.

    Techniques Used: Sample Prep, Transformation Assay

    rabbit monoclonal anti phospho p38  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho p38
    ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 <t>and</t> <t>anti-phospho</t> <t>p38</t> antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.
    Rabbit Monoclonal Anti Phospho P38, 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 "Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress"

    Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

    Journal: bioRxiv

    doi: 10.1101/2024.04.02.587770

    ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.
    Figure Legend Snippet: ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.

    Techniques Used: Immunoprecipitation, Western Blot, Quantitative RT-PCR, RNA Extraction, Expressing, Mutagenesis, Standard Deviation, Two Tailed Test, Activation Assay

    (A) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3 -mediated spindle checkpoint activation to activate MAPKs. Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-IP and time-course analysis on SAC or APC/C activation. (B) Immunoblot analysis of activation of MAPKs and Slp1 Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control. (C) Co-immunoprecipitation analysis of APC/C-MCC association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311 -arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1 Cdc20 were detected as in . Note that APC/C-MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures. (D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18 °C and KCl or caspofungin treatments. ( E, F ) Immunoblot analyses of Slp1 Cdc20 abundance and time-course analyses of SAC activation and inactivation efficiency in phosphorylation- and ubiquitylation-deficient mutants upon SAC activation and environmental stress.
    Figure Legend Snippet: (A) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3 -mediated spindle checkpoint activation to activate MAPKs. Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-IP and time-course analysis on SAC or APC/C activation. (B) Immunoblot analysis of activation of MAPKs and Slp1 Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control. (C) Co-immunoprecipitation analysis of APC/C-MCC association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311 -arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1 Cdc20 were detected as in . Note that APC/C-MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures. (D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18 °C and KCl or caspofungin treatments. ( E, F ) Immunoblot analyses of Slp1 Cdc20 abundance and time-course analyses of SAC activation and inactivation efficiency in phosphorylation- and ubiquitylation-deficient mutants upon SAC activation and environmental stress.

    Techniques Used: Activation Assay, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Cell Culture

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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 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 rabbit anti phospho p38 antibody
    Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of <t>p38</t> and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.
    Rabbit Anti Phospho P38 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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    86
    Cell Signaling Technology Inc rabbit anti phospho p38
    Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of <t>p38</t> and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.
    Rabbit Anti Phospho P38, 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 polyclonal anti phospho p38 mapk thr180 tyr182 antibody
    Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, <t>phospho-p38,</t> and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.
    Rabbit Polyclonal Anti 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 rabbit anti phospho p38 mapk t180 y182
    Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the <t>p38</t> pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).
    Rabbit Anti Phospho P38 Mapk T180 Y182, 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 monoclonal anti phospho p38
    ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 <t>and</t> <t>anti-phospho</t> <t>p38</t> antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.
    Rabbit Monoclonal Anti Phospho P38, 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:

    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

    Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of p38 and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.

    Journal: Neural Regeneration Research

    Article Title: Casein kinase-2 inhibition promotes retinal ganglion cell survival after acute intraocular pressure elevation

    doi: 10.4103/1673-5374.385310

    Figure Lengend Snippet: Western blotting analysis of the relative expression of MAPK signaling pathway components in the retina in the different intervention groups. The rats were subjected to IOP elevation, and then received the intravitreal injection of DMSO, the CK2 inhibitor TBB, the CK2 inhibitor DMAT, and the macrophage activator ZYM. Rats that were not subjected to IOP elevation and were intravitreally injected with DMSO served as intact controls. (A) The relative protein expression levels of p38 and p-p38 were examined by western blotting. (B) The relative protein expression levels of Erk p44/p42 and p-p44/p42 were detected by western blotting. The results are presented as histograms after normalization to β-actin or GAPDH. The data are presented as mean ± SD ( n = 5). * P < 0.05 (one-way analysis of variance among multiple groups followed by post hoc Bonferroni test). CK2: Casein kinase-2; DMAT: 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMSO: dimethylsulfoxide; Erk: extracellular-signal-regulated kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IOP: intraocular pressure; MAPK: mitogen-activated protein kinase; RGCs: retinal ganglion cells; TBB: 4,5,6,7-tetrabromo-2-azabenzimidazole; ZYM: zymosan.

    Article Snippet: Next, 50 μg of total protein was resolved by SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), transferred onto a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK), blocked with 5% skim milk in 0.05% Tween 20 (ICN, Erie, PA, USA) in Tris-buffered saline, and probed with rabbit anti-extracellular-signal-regulated kinase (Erk) p44/p42 antibody (1:1000, Cell Signaling Technology, Beverly, MA, USA, Cat# 4695, RRID: AB_390779), rabbit anti-phospho-Erk p44/p42 antibody (1:1000, Cell Signaling Technology, Cat# 8544, RRID: AB_11127856), rabbit anti-p38 antibody (1:1000, Cell Signaling Technology, Cat# 14451, RRID: AB_2798482), or rabbit anti-phospho-p38 antibody (1:1000, Cell Signaling Technology, Cat# 4511, RRID: AB_2139682) in 3% bovine serum albumin at 4°C overnight.

    Techniques: Western Blot, Expressing, Injection

    Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.

    Journal: iScience

    Article Title: Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells

    doi: 10.1016/j.isci.2024.109675

    Figure Lengend Snippet: Ganoderma lucidum (GL) extract treatment prevents the phosphorylation of proteins in the MAPK signaling pathways in corticotropin-releasing hormone (CRH)-related stress response (A) Dermal papilla cells (DPCs) and (B) outer root sheath cells (ORSCs) were treated with CRH (0.1 μM–0.5 μM) in the absence and the presence of GL extract (20 μg/mL) for 48 h. Cultured cells were harvested and western blotting was performed using antibodies against phospho-JNK, total-JNK, phospho- c -jun, total- c -jun, phospho-Erk1/2, total-Erk1/2, phospho-p38, and total-p38. Equal protein loading was confirmed based on total-JNK, total- c -jun, total-Erk1/2, and total-p38 levels. The graphs represent the quantification of the p -JNK/t-JNK, p- c -jun/c-jun, p -Erk1/2/t-Erk1/2, and p-p38/t-p38 protein expression. Data are presented as mean ± SD of five independent experiments. Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparisons test. ∗ p < 0.05, ∗∗ p < 0.01.

    Article Snippet: Rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibody, unconjugated , Cell Signaling Technology , Cat #9211, RRID: AB_331641.

    Techniques: Cell Culture, Western Blot, Expressing

    Journal: iScience

    Article Title: Ganoderma lucidum extract attenuates corticotropin-releasing hormone-induced cellular senescence in human hair follicle cells

    doi: 10.1016/j.isci.2024.109675

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-phospho-p38 MAPK (Thr180/Tyr182) antibody, unconjugated , Cell Signaling Technology , Cat #9211, RRID: AB_331641.

    Techniques: Recombinant, CCK-8 Assay, Staining, Protease Inhibitor, Saline, Blocking Assay, Ab Array, Software

    Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).

    Journal: Scientific Reports

    Article Title: Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

    doi: 10.1038/s41598-024-58619-1

    Figure Lengend Snippet: Investigation of phosphosites showing EGF-dependent up-regulation. ( A ) Scheme to filter peptides showing up-regulation by inhibitor treatment and summary of the numbers of the up-regulated phosphosites for each inhibitor ( B ) PCA of the log2-transformed abundance of phosphosites up-regulated by both 8 and 20 min of EGF incubation. ( C ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. Proteins with at least one connection with other proteins are included. ( D ) Illustration of the p38 pathway activated by EGFR. The figure was created using BioRender.com. ( E ) Sequence motif enrichment analysis of 15 residues surrounding the regulated phosphosites. ( F ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment. See also Fig. S5 for the heatmap containing all 80 peptides. ( G ) Western blotting analysis using the cell lysates of Hela cells pre-treated with the inhibitors (500 nM) for 15 min followed by 20 min incubation with EGF (20 nM).

    Article Snippet: Primary antibodies used were rabbit anti-EGFR (ab32198; Abcam), rabbit anti-phospho-EGFR (pY1045) (#2237; Cell Signaling Technology), mouse anti-phospho-EGFR (pY1068) (#2236; Cell Signaling Technology), rabbit anti-Erk1/2 (#4695; Cell Signaling Technology), mouse anti-phospho-Erk1/2 (T202/Y204) (#9106; Cell Signaling Technology), rabbit anti-Akt (#9272; Cell Signaling Technology) rabbit anti-phospho-Akt (S473) (#9271; Cell Signaling Technology), rabbit anti-p38 MAPK (#9212; Cell Signaling Technology), rabbit anti-phospho p38 MAPK (T180/Y182) (#9211; Cell Signaling Technology) and mouse anti-GAPDH (Abcam).

    Techniques: Transformation Assay, Incubation, Sequencing, Western Blot

    MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.

    Journal: Scientific Reports

    Article Title: Phosphoproteomic investigation of targets of protein phosphatases in EGFR signaling

    doi: 10.1038/s41598-024-58619-1

    Figure Lengend Snippet: MS-based phosphoproteomics of EGF signaling with treatment of PP2C inhibitor sanguinarine in presence of p38 inhibitor BIRB796. ( A ) Schematic illustration of MS-based phosphoproteomics sample preparation. The figure was created using BioRender.com. ( B ) STRING-based protein network of proteins showing sanguinarine-dependent up-regulation of its phosphorylation. ( C ) Heatmap showing the log2-transformed phosphosite abundance changes induced by inhibitor treatment.

    Article Snippet: Primary antibodies used were rabbit anti-EGFR (ab32198; Abcam), rabbit anti-phospho-EGFR (pY1045) (#2237; Cell Signaling Technology), mouse anti-phospho-EGFR (pY1068) (#2236; Cell Signaling Technology), rabbit anti-Erk1/2 (#4695; Cell Signaling Technology), mouse anti-phospho-Erk1/2 (T202/Y204) (#9106; Cell Signaling Technology), rabbit anti-Akt (#9272; Cell Signaling Technology) rabbit anti-phospho-Akt (S473) (#9271; Cell Signaling Technology), rabbit anti-p38 MAPK (#9212; Cell Signaling Technology), rabbit anti-phospho p38 MAPK (T180/Y182) (#9211; Cell Signaling Technology) and mouse anti-GAPDH (Abcam).

    Techniques: Sample Prep, Transformation Assay

    ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.

    Journal: bioRxiv

    Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

    doi: 10.1101/2024.04.02.587770

    Figure Lengend Snippet: ( A ) Co-immunoprecipitation analysis on MCC-APC/C association. Cells with indicated genotypes were grown at 30 °C to mid-log phase and arrested at 18 °C for 6 hours. Lid1-TAP was immunoprecipitated and associated Mad2, Mad3 and Slp1 Cdc20 were detected by immunoblotting. The amount of co-immunoprecipitated Mad2, Mad3 and Slp1 Cdc20 was quantified by being normalized to those of total immunoprecipitated Lid1 in each sample, with the relative ratio between Mad2-GFP plus Mad3-GFP or Slp1 Cdc20 and Lid1-TAP in wild-type sample set as 1.0. Blots are representative of three independent experiments. p values were calculated against wild-type cells. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; n.s., not significant. ( B, C ) Immunoblot analysis of Slp1 Cdc20 abundance in nda3-KM311 cells treated at 18 °C for 6 hr. Slp1 Cdc20 levels were quantified with the relative ratio between Slp1 Cdc20 and Cdc2 in wild-type strain set as 1.0. Phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P) in (A) were detected using anti-phospho p42/44 and anti-phospho p38 antibodies and represents activated CIP or SAP signaling, respectively. sty1-T97A was inactivated by 5μM 3-BrB-PP1. Blots shown are the representative of three independent experiments. p values were calculated against wild-type cells. ***, p <0.001; n.s., not significant. ( D ) RT-qPCR analysis of mRNA levels of slp1 + . Cells with indicated genotypes were grown and treated as in (A-C) before RNA extraction. The relative fold-change ( slp1 + / act1 + ) in mRNA expression was calculated with that in wild-type cells being normalized to 1.0. Note mRNA level of slp1 + in pek1 DD mutant is not decreased. Error bars indicate mean ±standard deviation of three independent experiments. Two-tailed unpaired t -test was used to derive p values. n.s, not significant. ( E ) Schematic summary of the negative effect of activated CIP and SAP signaling on APC/C activation based on primary phenotype characterization of pmk1Δ , sty1-T97A , pek1 DD and wis1 DD mutants.

    Article Snippet: The following antibodies used for immunoblot analyses were purchased from the indicated commercial sources and were used at the indicated dilution: peroxidase-anti-peroxidase (PAP) soluble complex (Sigma-Aldrich; P1291; 1:10,000); rabbit polyclonal anti-Myc (GeneScript; A00172-40; 1:2,000); mouse monoclonal anti-GFP (Beijing Ray Antibody Biotech; RM1008; 1:2,000); rat monoclonal anti-HA (Roche, Cat. No. 11 867 423 001; 1:2,000); rabbit polyclonal anti-Slp1 ( ) (1:500); rabbit polyclonal anti-phospho-p44/42 (detecting activated Pmk1) (Cell Signaling Technology; #9101; 1:1,000); rabbit monoclonal anti-phospho-p38 (Cell Signaling Technology; #4511; 1:1,000); rabbit polyclonal anti-PSTAIRE (detecting Cdc2) (Santa Cruz Biotechnology; sc-53; 1:1,000); rabbit monoclonal anti-Thiophosphate ester antibody (Abcam; ab239919; 1:5,000); goat anti-GST HRP-conjugated antibody (RRID:AB_771429; GE Healthcare; 1:10,000).

    Techniques: Immunoprecipitation, Western Blot, Quantitative RT-PCR, RNA Extraction, Expressing, Mutagenesis, Standard Deviation, Two Tailed Test, Activation Assay

    (A) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3 -mediated spindle checkpoint activation to activate MAPKs. Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-IP and time-course analysis on SAC or APC/C activation. (B) Immunoblot analysis of activation of MAPKs and Slp1 Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control. (C) Co-immunoprecipitation analysis of APC/C-MCC association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311 -arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1 Cdc20 were detected as in . Note that APC/C-MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures. (D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18 °C and KCl or caspofungin treatments. ( E, F ) Immunoblot analyses of Slp1 Cdc20 abundance and time-course analyses of SAC activation and inactivation efficiency in phosphorylation- and ubiquitylation-deficient mutants upon SAC activation and environmental stress.

    Journal: bioRxiv

    Article Title: Negative regulation of APC/C activation by MAPK-mediated attenuation of Cdc20 Slp1 under stress

    doi: 10.1101/2024.04.02.587770

    Figure Lengend Snippet: (A) Schematic depiction of the experimental design for treatment with KCl or caspofungin during nda3 -mediated spindle checkpoint activation to activate MAPKs. Samples were collected at indicated time points for subsequent analyses including immunoblotting, co-IP and time-course analysis on SAC or APC/C activation. (B) Immunoblot analysis of activation of MAPKs and Slp1 Cdc20 protein levels. Samples with or without indicated treatments were blotted with anti-phospho p42/44 and anti-phospho p38 antibodies as indicative of phosphorylated Pmk1 (Pmk1-P) or phosphorylated Sty1 (Sty1-P), respectively. Slp1 Cdc20 levels were detected with anti-Slp1 antibodies and anti-Cdc2 was used as loading control. (C) Co-immunoprecipitation analysis of APC/C-MCC association upon environmental stress. Lid1-TAP was immunoprecipitated from nda3-KM311 -arrested cells and associated Mad2-GFP, Mad3-GFP and Slp1 Cdc20 were detected as in . Note that APC/C-MCC association was disrupted when 0.6 M KCl was present during cell culturing or during immunoprecipitation procedures. (D) Time-course analyses of SAC activation and inactivation in nda3-KM311 cdc13-GFP strains with indicated genotypes after arrest at 18 °C and KCl or caspofungin treatments. ( E, F ) Immunoblot analyses of Slp1 Cdc20 abundance and time-course analyses of SAC activation and inactivation efficiency in phosphorylation- and ubiquitylation-deficient mutants upon SAC activation and environmental stress.

    Article Snippet: The following antibodies used for immunoblot analyses were purchased from the indicated commercial sources and were used at the indicated dilution: peroxidase-anti-peroxidase (PAP) soluble complex (Sigma-Aldrich; P1291; 1:10,000); rabbit polyclonal anti-Myc (GeneScript; A00172-40; 1:2,000); mouse monoclonal anti-GFP (Beijing Ray Antibody Biotech; RM1008; 1:2,000); rat monoclonal anti-HA (Roche, Cat. No. 11 867 423 001; 1:2,000); rabbit polyclonal anti-Slp1 ( ) (1:500); rabbit polyclonal anti-phospho-p44/42 (detecting activated Pmk1) (Cell Signaling Technology; #9101; 1:1,000); rabbit monoclonal anti-phospho-p38 (Cell Signaling Technology; #4511; 1:1,000); rabbit polyclonal anti-PSTAIRE (detecting Cdc2) (Santa Cruz Biotechnology; sc-53; 1:1,000); rabbit monoclonal anti-Thiophosphate ester antibody (Abcam; ab239919; 1:5,000); goat anti-GST HRP-conjugated antibody (RRID:AB_771429; GE Healthcare; 1:10,000).

    Techniques: Activation Assay, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation, Cell Culture