rabbit anti phospho p38 antibody  (Cell Signaling Technology Inc)


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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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    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

    phospho p38  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38
    CD133-containing MVs induce angiogenesis through the <t>p38</t> MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.
    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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    Images

    1) Product Images from "CD133-containing microvesicles promote colorectal cancer progression by inducing tumor angiogenesis"

    Article Title: CD133-containing microvesicles promote colorectal cancer progression by inducing tumor angiogenesis

    Journal: Heliyon

    doi: 10.1016/j.heliyon.2024.e29292

    CD133-containing MVs induce angiogenesis through the p38 MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.
    Figure Legend Snippet: CD133-containing MVs induce angiogenesis through the p38 MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.

    Techniques Used: Western Blot, Two Tailed Test

    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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    Images

    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

    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 anti phospho p38
    Phosphorylated <t>p38</t> level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001
    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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    Images

    1) Product Images from "Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons"

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    Journal: Molecular Autism

    doi: 10.1186/s13229-024-00595-4

    Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001
    Figure Legend Snippet: Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001

    Techniques Used: Western Blot, Two Tailed Test, Software

    p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm), p38α (green, 488 nm, FITC), and Neun (red, 561 nm, TRITC) in ARC. B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox and AAV9-Agrp-Cre mix injection to ARC in WT mice, green fluorescent protein (GFP, 488 nm) represents virus expression. C and D . Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice. E . The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger2 chamber. G . Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice. H . Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice. 10-week-old WT male mice were injected with virus. The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment. 11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment. Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.
    Figure Legend Snippet: p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm), p38α (green, 488 nm, FITC), and Neun (red, 561 nm, TRITC) in ARC. B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox and AAV9-Agrp-Cre mix injection to ARC in WT mice, green fluorescent protein (GFP, 488 nm) represents virus expression. C and D . Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice. E . The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger2 chamber. G . Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice. H . Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice. 10-week-old WT male mice were injected with virus. The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment. 11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment. Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Techniques Used: Over Expression, Immunofluorescence, Staining, Injection, Virus, Expressing, Activity Assay, Two Tailed Test, Software

    Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm). B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression. C and D , Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice. E . The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber. G . The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice. H . The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice. 10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox . The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment. 12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment. Home-Cage monitoring test was performed 4–6 weeks after AAV injection, three-chamber test was performed 8–10 weeks after AAV injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.
    Figure Legend Snippet: Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm). B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression. C and D , Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice. E . The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber. G . The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice. H . The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice. 10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox . The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment. 12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment. Home-Cage monitoring test was performed 4–6 weeks after AAV injection, three-chamber test was performed 8–10 weeks after AAV injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Techniques Used: Over Expression, Immunofluorescence, Staining, Generated, Injection, Virus, Expressing, Activity Assay, Two Tailed Test, Software

    Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . schematic of generating p38α AgRP-176/327 mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. D . the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. E . the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. F . the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber. p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group. Mice were subjected to Home-Cage monitoring test at the age of 8–9 weeks, and subjected to three-chamber test at the age of 9–10 weeks. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.
    Figure Legend Snippet: Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . schematic of generating p38α AgRP-176/327 mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. D . the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. E . the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. F . the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber. p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group. Mice were subjected to Home-Cage monitoring test at the age of 8–9 weeks, and subjected to three-chamber test at the age of 9–10 weeks. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Techniques Used: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

    Inactivated p38α in AgRP neurons ameliorates the autistic-like behaviors of Shank3 -/- mice. A . scheme of generating Shank3 -/- : p38α AgRP-180/182 mice and immunofluorescence staining of p-p38α(green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. D . the total distance in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. E . the activity time in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. F . the first phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger2 chamber. Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 male mice were subjected to Home-Cage monitoring test at the age of 8 weeks, and subjected to three-chamber test at the age of 14 weeks, 12 mice in Shank3 -/- : p38α AgRP-con and 19 mice in Shank3 -/- : p38α AgRP-180/182 group. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.
    Figure Legend Snippet: Inactivated p38α in AgRP neurons ameliorates the autistic-like behaviors of Shank3 -/- mice. A . scheme of generating Shank3 -/- : p38α AgRP-180/182 mice and immunofluorescence staining of p-p38α(green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. D . the total distance in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. E . the activity time in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. F . the first phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger2 chamber. Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 male mice were subjected to Home-Cage monitoring test at the age of 8 weeks, and subjected to three-chamber test at the age of 14 weeks, 12 mice in Shank3 -/- : p38α AgRP-con and 19 mice in Shank3 -/- : p38α AgRP-180/182 group. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Techniques Used: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

    phospho p38 mapk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho p38 mapk
    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 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

    anti phospho p38 mapk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho p38 mapk
    Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of <t>phospho-p38</t> versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.
    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 "Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors"

    Article Title: Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2024.12444

    Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.
    Figure Legend Snippet: Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

    Techniques Used: Activation Assay, Cell Culture, Incubation, SDS Page, Western Blot, Derivative Assay

    unlabeled rabbit monoclonal antibody phospho p38 mapk  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc unlabeled rabbit monoclonal antibody phospho p38 mapk
    Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with <t>p38</t> antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).
    Unlabeled Rabbit Monoclonal Antibody 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 "Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi ( Lates calcarifer )"

    Article Title: Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi ( Lates calcarifer )

    Journal: Comparative Immunology Reports

    doi: 10.1016/j.cirep.2024.200142

    Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with p38 antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).
    Figure Legend Snippet: Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with p38 antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).

    Techniques Used: Lysis, Concentration Assay, Microscopy, Staining, Isolation, Confocal Microscopy, Expressing, Fluorescence

    rabbit anti phospho p38 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho p38 antibody
    a , b Immunofluorescence staining for Mettl14 in peritoneal macrophages from p38α floxp/floxp ( p38α f/f ) or p38α floxp/floxp ; LysM-Cre ( p38α −/− ) mice stimulated with or without PGN for 2 h. Scale bar, 10 μm. Every point in b represents the number of Mettl14 foci in 30 cells. c Endogenous interaction of Mettl14 and <t>p38.</t> d Interaction of GST-p38 with GFP-METTL14 in vitro. e , f Endogenous interaction of Mettl14 and p38 in peritoneal macrophages infected with H37Rv for indicated times (MOI = 5). Relative gray intensity of p38 was shown in ( f ). g In vitro kinase assay of purified recombinant GST-p38 (active) with GFP-Mettl14-WT or GFP-Mettl14-T72A. h Mettl14-WT and Mettl14-T72A are subjected to in vitro kinase assay and then performed in vitro LLPS assay to test the ability of droplet formation. i IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). j IB and IP of HEK293T cells transfected with plasmids encoding FLAG-EsxB for 24 h and stimulated with TNF-α for indicated times. k – n Real-time fluorescence amplification curves and bar plot of the threshold cycle ( C T ) of qPCR showing SELECT results for detecting m 6 A3656 in Nox2 mRNA ( k ), qPCR analysis of Nox2 mRNA ( l ), Nox2 protein levels ( m ) and changes in the levels of ROS (DCF staining; green) ( n ) in peritoneal macrophages from p38α f/f or p38α −/− mice infected with H37Rv or H37Rv(ΔEsxB) for 2 h in k or 4–8 h in ( l – n) (MOI = 5). Rn is the raw fluorescence for the associated well normalized to the fluorescence of the passive reference dye (ROX). o IB analysis of protein levels from mouse macrophages infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. p IB analysis of iBMDM overexpression with Flag-p38 together with vector (-) or TAB1 or MKK6(E) or EsxB. q In vitro kinase assay of purified recombinant 6× His-p38 with TAB1 and EsxB. r IB analysis of mouse peritoneal macrophages treated with DMSO or SB203580 (10 μM) and infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. s IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). All of the immunoblot data are representative images from one of three independent experiments. Results in h were repeated twice independently. Results in b , f , k , l , n reflect the mean ± SEM from three independent biological experiments. Two-tailed unpaired Student’s t -test were used.
    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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    1) Product Images from "Mycobacterium tuberculosis inhibits METTL14-mediated m 6 A methylation of Nox2 mRNA and suppresses anti-TB immunity"

    Article Title: Mycobacterium tuberculosis inhibits METTL14-mediated m 6 A methylation of Nox2 mRNA and suppresses anti-TB immunity

    Journal: Cell Discovery

    doi: 10.1038/s41421-024-00653-4

    a , b Immunofluorescence staining for Mettl14 in peritoneal macrophages from p38α floxp/floxp ( p38α f/f ) or p38α floxp/floxp ; LysM-Cre ( p38α −/− ) mice stimulated with or without PGN for 2 h. Scale bar, 10 μm. Every point in b represents the number of Mettl14 foci in 30 cells. c Endogenous interaction of Mettl14 and p38. d Interaction of GST-p38 with GFP-METTL14 in vitro. e , f Endogenous interaction of Mettl14 and p38 in peritoneal macrophages infected with H37Rv for indicated times (MOI = 5). Relative gray intensity of p38 was shown in ( f ). g In vitro kinase assay of purified recombinant GST-p38 (active) with GFP-Mettl14-WT or GFP-Mettl14-T72A. h Mettl14-WT and Mettl14-T72A are subjected to in vitro kinase assay and then performed in vitro LLPS assay to test the ability of droplet formation. i IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). j IB and IP of HEK293T cells transfected with plasmids encoding FLAG-EsxB for 24 h and stimulated with TNF-α for indicated times. k – n Real-time fluorescence amplification curves and bar plot of the threshold cycle ( C T ) of qPCR showing SELECT results for detecting m 6 A3656 in Nox2 mRNA ( k ), qPCR analysis of Nox2 mRNA ( l ), Nox2 protein levels ( m ) and changes in the levels of ROS (DCF staining; green) ( n ) in peritoneal macrophages from p38α f/f or p38α −/− mice infected with H37Rv or H37Rv(ΔEsxB) for 2 h in k or 4–8 h in ( l – n) (MOI = 5). Rn is the raw fluorescence for the associated well normalized to the fluorescence of the passive reference dye (ROX). o IB analysis of protein levels from mouse macrophages infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. p IB analysis of iBMDM overexpression with Flag-p38 together with vector (-) or TAB1 or MKK6(E) or EsxB. q In vitro kinase assay of purified recombinant 6× His-p38 with TAB1 and EsxB. r IB analysis of mouse peritoneal macrophages treated with DMSO or SB203580 (10 μM) and infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. s IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). All of the immunoblot data are representative images from one of three independent experiments. Results in h were repeated twice independently. Results in b , f , k , l , n reflect the mean ± SEM from three independent biological experiments. Two-tailed unpaired Student’s t -test were used.
    Figure Legend Snippet: a , b Immunofluorescence staining for Mettl14 in peritoneal macrophages from p38α floxp/floxp ( p38α f/f ) or p38α floxp/floxp ; LysM-Cre ( p38α −/− ) mice stimulated with or without PGN for 2 h. Scale bar, 10 μm. Every point in b represents the number of Mettl14 foci in 30 cells. c Endogenous interaction of Mettl14 and p38. d Interaction of GST-p38 with GFP-METTL14 in vitro. e , f Endogenous interaction of Mettl14 and p38 in peritoneal macrophages infected with H37Rv for indicated times (MOI = 5). Relative gray intensity of p38 was shown in ( f ). g In vitro kinase assay of purified recombinant GST-p38 (active) with GFP-Mettl14-WT or GFP-Mettl14-T72A. h Mettl14-WT and Mettl14-T72A are subjected to in vitro kinase assay and then performed in vitro LLPS assay to test the ability of droplet formation. i IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). j IB and IP of HEK293T cells transfected with plasmids encoding FLAG-EsxB for 24 h and stimulated with TNF-α for indicated times. k – n Real-time fluorescence amplification curves and bar plot of the threshold cycle ( C T ) of qPCR showing SELECT results for detecting m 6 A3656 in Nox2 mRNA ( k ), qPCR analysis of Nox2 mRNA ( l ), Nox2 protein levels ( m ) and changes in the levels of ROS (DCF staining; green) ( n ) in peritoneal macrophages from p38α f/f or p38α −/− mice infected with H37Rv or H37Rv(ΔEsxB) for 2 h in k or 4–8 h in ( l – n) (MOI = 5). Rn is the raw fluorescence for the associated well normalized to the fluorescence of the passive reference dye (ROX). o IB analysis of protein levels from mouse macrophages infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. p IB analysis of iBMDM overexpression with Flag-p38 together with vector (-) or TAB1 or MKK6(E) or EsxB. q In vitro kinase assay of purified recombinant 6× His-p38 with TAB1 and EsxB. r IB analysis of mouse peritoneal macrophages treated with DMSO or SB203580 (10 μM) and infected with H37Rv or H37Rv(ΔEsxB) (MOI = 5) for indicated times. s IB and IP of mouse peritoneal macrophages infected with H37Rv or H37Rv(ΔEsxB) for indicated times (MOI = 5). All of the immunoblot data are representative images from one of three independent experiments. Results in h were repeated twice independently. Results in b , f , k , l , n reflect the mean ± SEM from three independent biological experiments. Two-tailed unpaired Student’s t -test were used.

    Techniques Used: Immunofluorescence, Staining, In Vitro, Infection, Kinase Assay, Purification, Recombinant, Transfection, Fluorescence, Amplification, Over Expression, Plasmid Preparation, Western Blot, Two Tailed Test

    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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    Structured Review

    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
    Average 86 stars, based on 1 article reviews
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    Images

    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:

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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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    Cell Signaling Technology Inc phospho p38
    CD133-containing MVs induce angiogenesis through the <t>p38</t> MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.
    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 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 anti phospho p38
    Phosphorylated <t>p38</t> level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001
    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
    Phosphorylated <t>p38</t> level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001
    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 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 anti phospho p38 mapk
    Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of <t>phospho-p38</t> versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.
    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 unlabeled rabbit monoclonal antibody phospho p38 mapk
    Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with <t>p38</t> antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).
    Unlabeled Rabbit Monoclonal Antibody Phospho P38 Mapk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p38 mapk thr180 tyr182 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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    Image Search Results


    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

    CD133-containing MVs induce angiogenesis through the p38 MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.

    Journal: Heliyon

    Article Title: CD133-containing microvesicles promote colorectal cancer progression by inducing tumor angiogenesis

    doi: 10.1016/j.heliyon.2024.e29292

    Figure Lengend Snippet: CD133-containing MVs induce angiogenesis through the p38 MAPK signaling pathway. (A) EA.hy926 cells were treated with shMock and shCD133 MVs for 16 h. Protein level was determined using western blotting. p-p38, phosphorylated p38; pAKT, phosphorylated AKT; pERK, phosphorylated ERK. Full-length blot images are shown in (B) Quantification of the p-p38 level. (C) EA.hy926 cells were pre-treated with SB203580 (20 μM) for 2 h and treated with shMock MVs in the presence of SB203580 for 13 h. Representative images of tube formation were shown. Scale bar = 100 μm. (D) Meshes and tubes were measured from five random fields in each well. Mean ± SEM of n = 3 independent experiments. All p values were obtained using an unpaired two-tailed Student's t -test. *p < 0.05, * *p < 0.01, ** *p < 0.001.

    Article Snippet: Mouse monoclonal antibodies recognizing phospho-p44/42 ERK and rabbit monoclonal hVEGFR2, p38, phospho-p38, and phospho-AKT antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Western Blot, Two Tailed Test

    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

    Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001

    Journal: Molecular Autism

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    doi: 10.1186/s13229-024-00595-4

    Figure Lengend Snippet: Phosphorylated p38 level in the hypothalamus is significantly enhanced in ASD mouse model. A . KEGG enrichment analysis of the transcriptome from Shank3 knockdown primary neurons (GSE47150). B . KEGG enrichment analysis for the hypothalamus of Shank3TG mice (GSE120609). C . Overlapping of signaling pathways of two datasets, the number represents the well-defined pathways, and non-defined pathways were excluded. D . Immunoblotting and intensity of phosphorylation of p38, ERK1/2, and JNK in hypothalamus from WT and Shank3 −/− mice ( n = 3 per group). E . Immunoblotting and intensity of phosphorylated p38, ERK1/2, and JNK in hypothalamus from WT, BTBR mice ( n = 3 per group). Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05, ** p < 0.01, *** p < 0.001

    Article Snippet: Tissue lysates were immunoblotted with antibodies: anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-phospho-p38 (p-p38; 1:1000, 4511, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (p-ERK; 1:1000, 4376, Cell Signaling Technology), anti-ERK (1:1000, 4695, Cell Signaling Technology), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; 1:1000, 9255, Cell Signaling Technology), anti-JNK (1:1000, 9252, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:7500, 60004-1-Ig, Proteintech), and Tubulin (1:5000, 11224-1-AP, Proteintech).

    Techniques: Western Blot, Two Tailed Test, Software

    p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm), p38α (green, 488 nm, FITC), and Neun (red, 561 nm, TRITC) in ARC. B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox and AAV9-Agrp-Cre mix injection to ARC in WT mice, green fluorescent protein (GFP, 488 nm) represents virus expression. C and D . Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice. E . The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger2 chamber. G . Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice. H . Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice. 10-week-old WT male mice were injected with virus. The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment. 11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment. Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Journal: Molecular Autism

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    doi: 10.1186/s13229-024-00595-4

    Figure Lengend Snippet: p38α overexpression in ARC is sufficient to elicit excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm), p38α (green, 488 nm, FITC), and Neun (red, 561 nm, TRITC) in ARC. B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox and AAV9-Agrp-Cre mix injection to ARC in WT mice, green fluorescent protein (GFP, 488 nm) represents virus expression. C and D . Groom time and bouts in 24 h of p38α ARC−con and p38α ARC−OE mice. E . The first phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α ARC−con and p38α ARC−OE mice in stranger2 chamber. G . Total distance in 24 h of p38α ARC−con and p38α ARC−OE mice. H . Activity time in 24 h of p38α ARC−con and p38α ARC−OE mice. 10-week-old WT male mice were injected with virus. The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α ARC−con group, 3 mice were excluded and 9 mice were included in the experiment. 11 injected mice in the p38α ARC−OE group, 3 mice were excluded and 8 mice were included in the experiment. Home-Cage monitoring test was performed 6 weeks after virus injection, three-chamber test was performed 8 weeks after virus injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Article Snippet: Tissue lysates were immunoblotted with antibodies: anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-phospho-p38 (p-p38; 1:1000, 4511, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (p-ERK; 1:1000, 4376, Cell Signaling Technology), anti-ERK (1:1000, 4695, Cell Signaling Technology), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; 1:1000, 9255, Cell Signaling Technology), anti-JNK (1:1000, 9252, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:7500, 60004-1-Ig, Proteintech), and Tubulin (1:5000, 11224-1-AP, Proteintech).

    Techniques: Over Expression, Immunofluorescence, Staining, Injection, Virus, Expressing, Activity Assay, Two Tailed Test, Software

    Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm). B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression. C and D , Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice. E . The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber. G . The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice. H . The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice. 10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox . The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment. 12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment. Home-Cage monitoring test was performed 4–6 weeks after AAV injection, three-chamber test was performed 8–10 weeks after AAV injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Journal: Molecular Autism

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    doi: 10.1186/s13229-024-00595-4

    Figure Lengend Snippet: Overexpression of p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . Immunofluorescence staining of DAPI (blue, 405 nm) and p38α (green, 488 nm, FITC) in ARC, AgRP-tomato mice were generated by mating Agrp-Cre mouse with Tomato-reporter mouse (red, 561 nm). B . Schematic and immunofluorescence image of the AAV9-p38α flox/flox stereotaxic injection to ARC in Agrp-Cre mice, GFP represents virus expression. C and D , Groom time and bouts in 24 h of p38α AgRP−con and p38α AgRP−OE mice. E . The first phase of the three-chamber test, the time of p38α AgRP−con and p38α AgRP−OE mice in stranger1 chamber. F . The second phase of the three-chamber test, the time of p38α AgRP−con and p38α ARC−OE mice in the stranger2 chamber. G . The total distance in 24 h of p38α AgRP−con and p38α AgRP−OE mice. H . The activity time in 24 h of p38α AgRP−con and p38α AgRP−OE mice. 10-week-old control and AgRP-Cre male mice were injected with AAV9-p38α flox/flox . The injection site is in the ARC area as an inclusion criterion. 12 injected mice in the p38α AgRP−con group, 4 mice were excluded and 8 mice were included in the experiment. 12 injected mice in the p38α AgRP−OE group, 3 mice were excluded and 9 mice were included in the experiment. Home-Cage monitoring test was performed 4–6 weeks after AAV injection, three-chamber test was performed 8–10 weeks after AAV injection. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Article Snippet: Tissue lysates were immunoblotted with antibodies: anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-phospho-p38 (p-p38; 1:1000, 4511, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (p-ERK; 1:1000, 4376, Cell Signaling Technology), anti-ERK (1:1000, 4695, Cell Signaling Technology), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; 1:1000, 9255, Cell Signaling Technology), anti-JNK (1:1000, 9252, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:7500, 60004-1-Ig, Proteintech), and Tubulin (1:5000, 11224-1-AP, Proteintech).

    Techniques: Over Expression, Immunofluorescence, Staining, Generated, Injection, Virus, Expressing, Activity Assay, Two Tailed Test, Software

    Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . schematic of generating p38α AgRP-176/327 mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. D . the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. E . the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. F . the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber. p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group. Mice were subjected to Home-Cage monitoring test at the age of 8–9 weeks, and subjected to three-chamber test at the age of 9–10 weeks. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Journal: Molecular Autism

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    doi: 10.1186/s13229-024-00595-4

    Figure Lengend Snippet: Activated p38α in AgRP neurons elicits excessive stereotypic behavior and impaired sociability in WT mice. A . schematic of generating p38α AgRP-176/327 mice and immunofluorescence staining of p-p38α (green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. D . the total distance in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. E . the activity time in 24 h of p38α AgRP-con and p38α AgRP-176/327 mice. F . the first phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of p38α AgRP-con and p38α AgRP-176/327 mice in stranger2 chamber. p38α AgRP-con and p38α AgRP-176/327 male mice were used for the experiment, 8 mice in p38α AgRP-con and 9 mice in p38α AgRP-176/327 group. Mice were subjected to Home-Cage monitoring test at the age of 8–9 weeks, and subjected to three-chamber test at the age of 9–10 weeks. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Article Snippet: Tissue lysates were immunoblotted with antibodies: anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-phospho-p38 (p-p38; 1:1000, 4511, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (p-ERK; 1:1000, 4376, Cell Signaling Technology), anti-ERK (1:1000, 4695, Cell Signaling Technology), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; 1:1000, 9255, Cell Signaling Technology), anti-JNK (1:1000, 9252, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:7500, 60004-1-Ig, Proteintech), and Tubulin (1:5000, 11224-1-AP, Proteintech).

    Techniques: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

    Inactivated p38α in AgRP neurons ameliorates the autistic-like behaviors of Shank3 -/- mice. A . scheme of generating Shank3 -/- : p38α AgRP-180/182 mice and immunofluorescence staining of p-p38α(green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. D . the total distance in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. E . the activity time in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. F . the first phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger2 chamber. Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 male mice were subjected to Home-Cage monitoring test at the age of 8 weeks, and subjected to three-chamber test at the age of 14 weeks, 12 mice in Shank3 -/- : p38α AgRP-con and 19 mice in Shank3 -/- : p38α AgRP-180/182 group. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Journal: Molecular Autism

    Article Title: Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons

    doi: 10.1186/s13229-024-00595-4

    Figure Lengend Snippet: Inactivated p38α in AgRP neurons ameliorates the autistic-like behaviors of Shank3 -/- mice. A . scheme of generating Shank3 -/- : p38α AgRP-180/182 mice and immunofluorescence staining of p-p38α(green, 488 nm, FITC). B and C , the groom time and bouts in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. D . the total distance in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. E . the activity time in 24 h of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice. F . the first phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger1 chamber. G . the second phase of the three-chamber test, the time of Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 mice in stranger2 chamber. Shank3 -/- : p38α AgRP-con and Shank3 -/- : p38α AgRP-180/182 male mice were subjected to Home-Cage monitoring test at the age of 8 weeks, and subjected to three-chamber test at the age of 14 weeks, 12 mice in Shank3 -/- : p38α AgRP-con and 19 mice in Shank3 -/- : p38α AgRP-180/182 group. Statistical analysis: data were analyzed using unpaired two-tailed Student’s t -test (Prism9, GraphPad Software Inc.). Data were represented as Mean ± SD. Significance levels are indicated with * p < 0.05.

    Article Snippet: Tissue lysates were immunoblotted with antibodies: anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-phospho-p38 (p-p38; 1:1000, 4511, Cell Signaling Technology), anti-phospho-extracellular signal-regulated kinase (p-ERK; 1:1000, 4376, Cell Signaling Technology), anti-ERK (1:1000, 4695, Cell Signaling Technology), anti-phospho-c-Jun NH2-terminal kinase (p-JNK; 1:1000, 9255, Cell Signaling Technology), anti-JNK (1:1000, 9252, Cell Signaling Technology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:7500, 60004-1-Ig, Proteintech), and Tubulin (1:5000, 11224-1-AP, Proteintech).

    Techniques: Immunofluorescence, Staining, Activity Assay, Two Tailed Test, Software

    ( 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

    Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Predominant control of PDGF/PDGF receptor signaling in the migration and proliferation of human adipose‑derived stem cells under culture conditions with a combination of growth factors

    doi: 10.3892/etm.2024.12444

    Figure Lengend Snippet: Activation of growth factor receptors and signal enzymes in growth factor-treated hASCs. hASCs were cultured in DMEM containing 10% FBS, followed by starvation for 16 h. The cells were then incubated in DMEM containing PDGF-BB (20 ng/ml), VEGF (1 ng/ml), HGF (1 ng/ml), PDGF-BB (20 ng/ml)/VEGF (1 ng/ml) or PDGF-BB (20 ng/ml)/HGF (1 ng/ml) for 20 min. The cells then were washed, collected and lysed. Next, cellular proteins were analyzed by SDS-PAGE using 4-15% gels, followed by (A) immunoblotting with the indicated primary antibodies. (B) Ratio of phospho-PDGFRb versus total PDGFRb, (C) ratio of phospho-VEGFR2 versus total VEGFR2, (D) ratio of phospho-c-Met versus total c-Met, (E) ratio of phospho-ERK1/2 versus total ERK1/2 and (F) ratio of phospho-p38 versus total p38 were calculated. Data are presented as the mean ± SD (n=3). ** P<0.01 vs. control. hASCs, human adipose-derived stem cells; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; HGF, hepatocyte growth factor.

    Article Snippet: After blocking with Blocking One-P reagent (cat. no. 05999-84; Nacalai Tesque, Inc.) for 60 min at room temperature, the membranes were incubated overnight at 4˚C with the following primary antibodies: Anti-phospho-Erk1/2 (1:1,000; cat. no. #4370; Cell Signaling Technology, Inc.), anti-Erk1/2 (1:1,000; cat. no. #4695; Cell Signaling Technology, Inc.), anti-phospho-PDGFRb (1:1,000; cat. no. GTX133525; GeneTex, Inc.), anti-PDGFRb (1:1,000; cat. no. 134491AP; Proteintech Group, Inc.), anti-phospho-c-Met (1:1,000; cat. no. 600401989S; Rockland Immunochemicals Inc.), anti-c-Met (1:1,000; cat. no. GTX631992; GeneTex, Inc.), anti-phospho-VEGFR2 (1:1,000; cat. no. CSBPA000703; Cusabio Technology, LLC), anti-VEGFR2 (1:1,000; cat. no. CSBPA008334; Cusabio Technology, LLC), anti-phospho-p38 MAPK (1:1,000; cat. no. #4511; Cell Signaling Technology, Inc.), anti-p38 MAPK (1:1,000; cat. no. #8690; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. #4970; Cell Signaling Technology, Inc.).

    Techniques: Activation Assay, Cell Culture, Incubation, SDS Page, Western Blot, Derivative Assay

    Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with p38 antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).

    Journal: Comparative Immunology Reports

    Article Title: Development and validation of a flow cytometry method to examine circulating leukocyte subpopulations in barramundi ( Lates calcarifer )

    doi: 10.1016/j.cirep.2024.200142

    Figure Lengend Snippet: Calibration of hypotonic lysis of RBCs, fixation of leukocytes and FACS analysis. (A) Leukocyte concentration and viability after hypotonic lysis of erythrocytes, conducted for different time periods. (B) Number of live cells from fishes S1, S2, S3 and S4 following different numbers of washes. First and second wash were used to isolate leukocytes from RBCs; third and fourth wash were used to clean the cells after the use of fixative (Cytofix). Samples were analyzed under the microscope using a hemocytometer. (C–E) Density plots of Hoechst- and PI-stained cells to detect live/dead cells at the different steps of leukocyte isolation. (F–I) FACS analyses of leukocytes after RBC lysis. (F) After a single wash post-lysis and fixation, showing both RBC debris and leukocyte populations. (G) After two post-lysis washes, fixation, and PI gating to obtain only the clear leukocyte population without the RBC debris. (H) Leukocytes were gated and sorted for further analysis by confocal microscopy. (I) Confocal microscopy images of the gated populations (bar = 10 µm). FCS – forward scatter; SSC – side scatter. (J, K) Fixed leukocytes stained with p38 antibody; second antibody (2nd Ab) conjugated to Alexa Fluor 488 was used as a control. Data present the analysis of leukocytes from 5 fish. (J) Percent expression of p38 and 2nd Ab. (K) Expression levels of p38 and 2nd Ab in units of geometric mean fluorescence intensity (MFI).

    Article Snippet: For p38-staining, cells were incubated with primary unlabeled rabbit monoclonal antibody phospho-p38 MAPK, clone D3F9 (Cell Signaling Technology, USA) diluted 1:200 in BD Perm/Wash buffer and 200 µL was added to each well, followed by incubation at 4 °C for 30 min.

    Techniques: Lysis, Concentration Assay, Microscopy, Staining, Isolation, Confocal Microscopy, Expressing, Fluorescence

    Journal: eLife

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

    doi: 10.7554/eLife.91060

    Figure Lengend Snippet:

    Article Snippet: Antibody , Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP Rabbit mAb (Rabbit Monoclonal) , Cell Signaling Technology , Cat# 4511 , WB (1:1000).

    Techniques: