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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    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 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
    https://www.bioz.com/result/rabbit monoclonal anti phospho p38/product/Cell Signaling Technology Inc
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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

    phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab
    Phospho P38 Mapk Thr180 Tyr182 D3f9 Xp 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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    phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab

    Phospho P38 Mapk Thr180 Tyr182 D3f9 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab/product/Cell Signaling Technology Inc
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    1) Product Images from "Intermittent fasting promotes type 3 innate lymphoid cells secreting IL-22 contributing to the beigeing of white adipose tissue"

    Article Title: Intermittent fasting promotes type 3 innate lymphoid cells secreting IL-22 contributing to the beigeing of white adipose tissue

    Journal: eLife

    doi: 10.7554/eLife.91060


    Figure Legend Snippet:

    Techniques Used:

    rabbit monoclonal anti phospho p38  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit monoclonal anti phospho p38
    ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, <t>p-p38,</t> p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.
    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 "Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection"

    Article Title: Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection

    Journal: EMBO Reports

    doi: 10.1038/s44319-024-00072-2

    ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.
    Figure Legend Snippet: ( A ) Presence of type I (ALK1, ALK2, ALK3) and type II (BMPR2) receptors in HFF-1 and 293T was verified by immunoblotting with the respective antibodies. Detection of GAPDH protein served as loading control. ( B ) Relative transcript levels of type I (ALK1, ALK2, ALK3, ALK6) and type II (BMPR2, ACVR2A, ACVR2B) BMP receptors in 293T were determined by RT-qPCR. ( C ) 293T were co-transfected with expression plasmids for the BRE-Luciferase reporter and a Renilla luciferase normalization control (EF1α-Renilla). 24 h post transfection, 293T were either stimulated with BMP9 (3 nM), or BMP9 (3 nM) incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml or 5 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( D ) 293T were co-transfected as in ( B ). 24 h post transfection, 293 T were either stimulated with BMP4 (18 nM), BMP6 (18 nM), BMP9 (3 nM), BMP15 (18 nM), or Activin B (4 nM), or with the ligands incubated for 15 min at RT with an α-BMP9 antibody (1 µg/ml) for 16 h, followed by a dual-luciferase assay readout. ( E ) HFF-1 were either mock, DMSO, Ruxolitinib (10 µM) or DMH1 (10 µM) treated for 2 h, followed by stimulation with IFNβ (5 ng/ml) or BMP4 (18 nM) for 2 h. Cells were lysed and lysates were subjected to immunoblot analysis with p-STAT1, STAT1, p-SMAD1/5/9, SMAD1, p-p38, p38, p-p44/42, p44/42, and Calnexin-specific antibodies. Data information: ( A – E ) Experiments were performed two independent times, one representative is shown. Luciferase fold induction was calculated by dividing Renilla-normalized values from stimulated samples by the corresponding values from unstimulated samples.

    Techniques Used: Western Blot, Quantitative RT-PCR, Transfection, Expressing, Luciferase, Incubation

    rrid ab 823588 rabbit mab anti phospho p38 mapk d3f9 cell signaling 4511t  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rrid ab 823588 rabbit mab anti phospho p38 mapk d3f9 cell signaling 4511t
    Rrid Ab 823588 Rabbit Mab Anti Phospho P38 Mapk D3f9 Cell Signaling 4511t, 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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    phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab
    Phospho P38 Mapk Thr180 Tyr182 D3f9 Xp 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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    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
    (D-Ala 2 )GIP impaired LPS-induced MAPKs pathway activation in peritoneal macrophages in vitro. Peritoneal macrophages were starved for 6 h and then treated with LPS alone or LPS+(D-Ala 2 )GIP for 0, 5, 15, and 30 min. Proteins were isolated and used in Western blotting to quantify <t>phospho-p38/ERK/JNK</t> and β-actin. ( A ) Band images of phospho-p38/ERK/JNK and β-actin. ( B ) Quantification of phospho-p38 relative to β-actin. ( C ) Quantification of phospho-ERK relative to β-actin. ( D ) Quantification of phospho-JNK relative to β-actin. Data are presented as mean values ± SD. Paired t -test was adopted to evaluate the significance of variances ( n = 3; * p < 0.05).
    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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    1) Product Images from "(D-Ala 2 )GIP Inhibits Inflammatory Bone Resorption by Suppressing TNF-α and RANKL Expression and Directly Impeding Osteoclast Formation"

    Article Title: (D-Ala 2 )GIP Inhibits Inflammatory Bone Resorption by Suppressing TNF-α and RANKL Expression and Directly Impeding Osteoclast Formation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms25052555

    (D-Ala 2 )GIP impaired LPS-induced MAPKs pathway activation in peritoneal macrophages in vitro. Peritoneal macrophages were starved for 6 h and then treated with LPS alone or LPS+(D-Ala 2 )GIP for 0, 5, 15, and 30 min. Proteins were isolated and used in Western blotting to quantify phospho-p38/ERK/JNK and β-actin. ( A ) Band images of phospho-p38/ERK/JNK and β-actin. ( B ) Quantification of phospho-p38 relative to β-actin. ( C ) Quantification of phospho-ERK relative to β-actin. ( D ) Quantification of phospho-JNK relative to β-actin. Data are presented as mean values ± SD. Paired t -test was adopted to evaluate the significance of variances ( n = 3; * p < 0.05).
    Figure Legend Snippet: (D-Ala 2 )GIP impaired LPS-induced MAPKs pathway activation in peritoneal macrophages in vitro. Peritoneal macrophages were starved for 6 h and then treated with LPS alone or LPS+(D-Ala 2 )GIP for 0, 5, 15, and 30 min. Proteins were isolated and used in Western blotting to quantify phospho-p38/ERK/JNK and β-actin. ( A ) Band images of phospho-p38/ERK/JNK and β-actin. ( B ) Quantification of phospho-p38 relative to β-actin. ( C ) Quantification of phospho-ERK relative to β-actin. ( D ) Quantification of phospho-JNK relative to β-actin. Data are presented as mean values ± SD. Paired t -test was adopted to evaluate the significance of variances ( n = 3; * p < 0.05).

    Techniques Used: Activation Assay, In Vitro, Isolation, Western Blot

    (D-Ala 2 )GIP impaired LPS-induced MAPKs pathway activation in osteoblasts in vitro. Osteoblasts were starved for 6 h and then treated with LPS alone or LPS+(D-Ala 2 )GIP for 0, 5, 15, 30 min. Proteins were isolated and used in Western blotting to quantify phospho-p38/ERK/JNK and β-actin. ( A ) Band images of phospho-p38/ERK/JNK and β-actin. ( B ) Quantification of phospho-p38 relative to β-actin. ( C ) Quantification of phospho-ERK relative to β-actin. ( D ) Quantification of phospho-JNK relative to β-actin. Data are presented as mean values ± SD. Paired t -test was adopted to evaluate the significance of variances ( n = 3; * p < 0.05, ** p < 0.01).
    Figure Legend Snippet: (D-Ala 2 )GIP impaired LPS-induced MAPKs pathway activation in osteoblasts in vitro. Osteoblasts were starved for 6 h and then treated with LPS alone or LPS+(D-Ala 2 )GIP for 0, 5, 15, 30 min. Proteins were isolated and used in Western blotting to quantify phospho-p38/ERK/JNK and β-actin. ( A ) Band images of phospho-p38/ERK/JNK and β-actin. ( B ) Quantification of phospho-p38 relative to β-actin. ( C ) Quantification of phospho-ERK relative to β-actin. ( D ) Quantification of phospho-JNK relative to β-actin. Data are presented as mean values ± SD. Paired t -test was adopted to evaluate the significance of variances ( n = 3; * p < 0.05, ** p < 0.01).

    Techniques Used: Activation Assay, In Vitro, Isolation, Western Blot

    phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab  (Cell Signaling Technology Inc)


    Bioz Manufacturer Symbol Cell Signaling Technology Inc manufactures this product  
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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab
    Phospho P38 Mapk Thr180 Tyr182 D3f9 Xp Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab/product/Cell Signaling Technology Inc
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    anti phospho p38 mapk thr180 tyr182 d3f9 rabbit mab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho p38 mapk thr180 tyr182 d3f9 rabbit mab
    Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or <t>p38</t> in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.
    Anti Phospho P38 Mapk Thr180 Tyr182 D3f9 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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    1) Product Images from "Oncogenic K‐Ras G12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts"

    Article Title: Oncogenic K‐Ras G12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.13775

    Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or p38 in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.
    Figure Legend Snippet: Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or p38 in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.

    Techniques Used: Expressing, Cell Culture, Cell Counting, Western Blot

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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 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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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 d3f9 xp rabbit mab
    ( 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.
    Phospho P38 Mapk Thr180 Tyr182 D3f9 Xp 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 rrid ab 823588 rabbit mab anti phospho p38 mapk d3f9 cell signaling 4511t
    ( 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.
    Rrid Ab 823588 Rabbit Mab Anti Phospho P38 Mapk D3f9 Cell Signaling 4511t, 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/rrid ab 823588 rabbit mab anti phospho p38 mapk d3f9 cell signaling 4511t/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    rrid ab 823588 rabbit mab anti phospho p38 mapk d3f9 cell signaling 4511t - by Bioz Stars, 2024-05
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    Cell Signaling Technology Inc anti phospho p38 mapk thr180 tyr182 d3f9 rabbit mab
    Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or <t>p38</t> in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.
    Anti Phospho P38 Mapk Thr180 Tyr182 D3f9 Rabbit Mab, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho p38 mapk thr180 tyr182 d3f9 rabbit mab/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti phospho p38 mapk thr180 tyr182 d3f9 rabbit mab - by Bioz Stars, 2024-05
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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 ) 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

    Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or p38 in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.

    Journal: FEBS Open Bio

    Article Title: Oncogenic K‐Ras G12V cannot overcome proliferation failure caused by loss of Ppp6c in mouse embryonic fibroblasts

    doi: 10.1002/2211-5463.13775

    Figure Lengend Snippet: Loss of Ppp6c greatly reduces proliferation of K‐Ras G12V ‐expressing MEFs and does not markedly activate ERK1/2, JNK, or p38 in these cells. (A) Five hundred cells were seeded per well into a 96‐well plate and cultured for up to 5 days. Cell numbers were determined using a Cell Counting Kit‐8. Values are expressed relative to the OD450 values on Day 1. ●, 4HT‐untreated CreERT‐Ppp6c fl/fl MEFs; ○, 4HT‐treated CreERT‐Ppp6c fl/fl MEFs; ■, 4HT‐untreated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs; and □, 4HT‐treated CreERT‐Ppp6c fl/fl ‐K‐Ras G12V MEFs. For MEFs, n = 3 for 4HT + and 4HT − . For K‐Ras G12V ‐expressing MEFs, n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. *** P < 0.001 by the Student's t test. (B) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in K‐Ras G12V ‐expressing MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (C) The results from (B) were quantified using imagej . p‐MAPK values were divided by MAPK values and the resulting values were plotted, with the average value in K‐Ras G12V ‐expressing MEFs without 4HT set to 1. White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. n.s., P > 0.05 by the Student's t test. (D) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation levels of MAPKs in CreERT‐Ppp6c fl/fl MEFs. 4HT + and 4HT − represent with and without 4HT, respectively. (E) The results from (D) were quantified using ImageJ as in (C). White circles in the graph indicate values for each sample. n = 3 for 4HT + and 4HT − . Data are presented as mean ± SD of three independent experiments. * P < 0.05 by the Student's t test. (F) Western blotting was performed to examine the effect of Ppp6c deficiency on the phosphorylation level of Akt in CreERT‐Ppp6c fl/fl MEFs with and without K‐Ras G12V . 4HT + and 4HT − represent with and without 4HT, respectively.

    Article Snippet: Other antibodies were purchased as follows: anti‐p44/42 mitogen‐activated protein kinase (MAPK) (237F5) rabbit monoclonal antibody (mAb) (#4695, CST, Danvers, MA, USA), anti‐SAPK/JNK antibody (#9252, CST), anti‐p38 MAPK (D13E1) XP rabbit mAb (#8690, CST), anti‐Akt antibody (#9272, CST), anti‐phospho‐p44/42 MAPK (Thr202/Tyr204) (D13, 14, 4E) XP rabbit mAb (#4370, CST), anti‐phospho‐p38 MAPK (Thr180/Tyr182) (D3F9) rabbit mAb (#4695, CST), anti‐phospho‐SAPK/JNK (Thr183/Thr185) (81E11) rabbit mAb (#4695, CST), anti‐phospho‐Akt (Ser473) (D9E) XP ® Rabbit mAb (#4060, CST), anti‐Ras (27H5) rabbit mAb (#3339S, CST), and anti‐β‐actin mouse mAb (AC‐15) (#A5441, Sigma‐Aldrich).

    Techniques: Expressing, Cell Culture, Cell Counting, Western Blot