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R&D Systems active human recombinant p38α mapk

The effect of <t>p38</t> activity on FRS2. ( a – c ) FGF1-induced electrophoretic mobility shift of FRS2. Serum-starved ( a , c ) NIH3T3 and ( b ) U2OS-R1 were pretreated for 20 min with or without MEK1/2 inhibitors (20 µM U0126, 1 µM SL327), p38 inhibitor (5 µM SB203580), and p38 activator (10 µM anisomycin), and then stimulated with the growth factor in the presence of heparin (10 U/mL) for 15 min. Cells were lysed, and the cellular material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( a , b ) or Phos-Tag SDS-PAGE ( c ) and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 or anti-tubulin as a loading control. ( d ) In vitro phosphorylation of FRS2 by p38α kinase. <t>Recombinant,</t> active kinase p38α and partial recombinant fusion protein FRS2α with GST tag were incubated with (γ- 33 P) ATP in reaction buffer at 30 °C for 30 min in the presence or absence of 5 µM SB203580. Erk1 and Erk2 kinases served as positive controls. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). Representative experiments are shown, for ( a ) and ( b ) n = 4, and for ( c ) and ( d ) n = 2.

Active Human Recombinant P38α Mapk, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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active human recombinant p38α mapk - by Bioz Stars, 2026-07

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1) Product Images from "Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling"

Article Title: Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20081826

The effect of p38 activity on FRS2. ( a – c ) FGF1-induced electrophoretic mobility shift of FRS2. Serum-starved ( a , c ) NIH3T3 and ( b ) U2OS-R1 were pretreated for 20 min with or without MEK1/2 inhibitors (20 µM U0126, 1 µM SL327), p38 inhibitor (5 µM SB203580), and p38 activator (10 µM anisomycin), and then stimulated with the growth factor in the presence of heparin (10 U/mL) for 15 min. Cells were lysed, and the cellular material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( a , b ) or Phos-Tag SDS-PAGE ( c ) and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 or anti-tubulin as a loading control. ( d ) In vitro phosphorylation of FRS2 by p38α kinase. Recombinant, active kinase p38α and partial recombinant fusion protein FRS2α with GST tag were incubated with (γ- 33 P) ATP in reaction buffer at 30 °C for 30 min in the presence or absence of 5 µM SB203580. Erk1 and Erk2 kinases served as positive controls. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). Representative experiments are shown, for ( a ) and ( b ) n = 4, and for ( c ) and ( d ) n = 2.

Figure Legend Snippet: The effect of p38 activity on FRS2. ( a – c ) FGF1-induced electrophoretic mobility shift of FRS2. Serum-starved ( a , c ) NIH3T3 and ( b ) U2OS-R1 were pretreated for 20 min with or without MEK1/2 inhibitors (20 µM U0126, 1 µM SL327), p38 inhibitor (5 µM SB203580), and p38 activator (10 µM anisomycin), and then stimulated with the growth factor in the presence of heparin (10 U/mL) for 15 min. Cells were lysed, and the cellular material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( a , b ) or Phos-Tag SDS-PAGE ( c ) and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 or anti-tubulin as a loading control. ( d ) In vitro phosphorylation of FRS2 by p38α kinase. Recombinant, active kinase p38α and partial recombinant fusion protein FRS2α with GST tag were incubated with (γ- 33 P) ATP in reaction buffer at 30 °C for 30 min in the presence or absence of 5 µM SB203580. Erk1 and Erk2 kinases served as positive controls. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). Representative experiments are shown, for ( a ) and ( b ) n = 4, and for ( c ) and ( d ) n = 2.

Techniques Used: Activity Assay, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Control, In Vitro, Phospho-proteomics, Recombinant, Incubation, Autoradiography, Membrane, Staining

$The crosstalk between p38 and Erk1/2 in downregulation of FGF1-induced signaling. Serum-starved ( a ) NIH3T3 and ( b ) U2OS-R1 cells were pretreated for 30 min with or without 20 µM U0126, 5 µM SB203580 and 10 µM anisomycin, and then stimulated with the growth factor in the presence of heparin (10 U/mL) and brefeldin A (2 μg/mL) for different time points. Cells were lysed, and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 as a loading control. Anti-phospho-Erk1/2 (p-Erk1/2) and anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38) were used to control the effect of U0126 (MEK inhibitor) and anisomycin (p38 activator). Representative experiments are shown, n = 3. The graphs present quantification of bands from panel b corresponding to phospho-FGFR1 (Tyr653/Tyr654) and phospho-FRS2 (Tyr196) normalized to loading control (Hsp90) and expressed as a fraction of the maximal response in the absence of inhibitor. Data are means ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.$
Figure Legend Snippet: The crosstalk between p38 and Erk1/2 in downregulation of FGF1-induced signaling. Serum-starved ( a ) NIH3T3 and ( b ) U2OS-R1 cells were pretreated for 30 min with or without 20 µM U0126, 5 µM SB203580 and 10 µM anisomycin, and then stimulated with the growth factor in the presence of heparin (10 U/mL) and brefeldin A (2 μg/mL) for different time points. Cells were lysed, and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 as a loading control. Anti-phospho-Erk1/2 (p-Erk1/2) and anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38) were used to control the effect of U0126 (MEK inhibitor) and anisomycin (p38 activator). Representative experiments are shown, n = 3. The graphs present quantification of bands from panel b corresponding to phospho-FGFR1 (Tyr653/Tyr654) and phospho-FRS2 (Tyr196) normalized to loading control (Hsp90) and expressed as a fraction of the maximal response in the absence of inhibitor. Data are means ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques Used: SDS Page, Western Blot, Control

The effect of p38 kinase activity on Erk1/2 activity and FGF1-induced signaling. ( a ) The effect of p38 specific inhibitor on Erk1/2 activity. Serum-starved NIH3T3 cells were treated with increasing concentration of the specific p38 kinase inhibitor SB203580 for 30 min. Then, the cells were lysed and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-Erk1/2 (p-Erk1/2), anti-Erk1/2, and anti-Hsp90 antibodies as a loading control. A representative experiment is shown, n = 4. The graph presents quantification of bands corresponding to phospho-Erk1/2 (p-Erk1/2) normalized to loading control (Hsp90) and expressed as a fold of change in comparison with untreated control. Data are means ± SD of four independent experiments; ** p < 0.01, *** p < 0.001. ( b ) Schematic representation of synergistic effect of p38 and Erk1/2 in the downregulation of FGF1-induced signaling through FRS2. FGF1-induced tyrosine phosphorylation of FGFR1 leads to the activation of FRS2 followed by GRB2/SOS-mediated activation of RAS and MAP kinases (Erk1/2 and p38). Activated Erks are supported by p38 in phosphorylation of FRS2 (red arrows), constituting a negative feedback loop that results in reduced tyrosine phosphorylation of FRS2 and consequent attenuation of FGFR signaling. Grey dashed line represents functional cross-talk between Erks and p38.

Figure Legend Snippet: The effect of p38 kinase activity on Erk1/2 activity and FGF1-induced signaling. ( a ) The effect of p38 specific inhibitor on Erk1/2 activity. Serum-starved NIH3T3 cells were treated with increasing concentration of the specific p38 kinase inhibitor SB203580 for 30 min. Then, the cells were lysed and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-Erk1/2 (p-Erk1/2), anti-Erk1/2, and anti-Hsp90 antibodies as a loading control. A representative experiment is shown, n = 4. The graph presents quantification of bands corresponding to phospho-Erk1/2 (p-Erk1/2) normalized to loading control (Hsp90) and expressed as a fold of change in comparison with untreated control. Data are means ± SD of four independent experiments; ** p < 0.01, *** p < 0.001. ( b ) Schematic representation of synergistic effect of p38 and Erk1/2 in the downregulation of FGF1-induced signaling through FRS2. FGF1-induced tyrosine phosphorylation of FGFR1 leads to the activation of FRS2 followed by GRB2/SOS-mediated activation of RAS and MAP kinases (Erk1/2 and p38). Activated Erks are supported by p38 in phosphorylation of FRS2 (red arrows), constituting a negative feedback loop that results in reduced tyrosine phosphorylation of FRS2 and consequent attenuation of FGFR signaling. Grey dashed line represents functional cross-talk between Erks and p38.

Techniques Used: Activity Assay, Concentration Assay, SDS Page, Western Blot, Control, Comparison, Phospho-proteomics, Activation Assay, Functional Assay

Image Search Results

Fig. 4 e Phosphorylation of signaling molecules in response to leptin by asthmatic and Healthy Control (HC) ﬁbroblasts. (A) Normal lung ﬁbroblasts (NHLFs) were seeded in 24-well plates and cultured for 1 h with the following inhibitors of intracellular signaling: AG490 (AG), 100 mM; SB203580 (SB), 10 mM; and U0126 (U), 30 mM. They were then stimulated with 10 mM leptin for 24 h. CCL11/eotaxin and CCL2/MCP-1 levels in the supernatant were determined using Cytometric Bead Array (CBA). Error bars represent the standard error of the mean (SEM) (n ¼ 5e6). *p < 0.05, **p < 0.01. (B) NHLFs were stimulated with 10 mM leptin for 0, 1, 3, 5, 10, or 60 min. The lysates were analyzed by western blotting with anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, or anti-GAPDH antibodies (left panels). Data are representative of 3e4 independent replicates. Quantitative analysis was achieved by densitometry (right panels), and the data are presented as the ratios indicated in the ﬁgure. Error bars represent the SEM (n ¼ 3e4). *p < 0.05, ***p < 0.001 vs.

Journal: Respiratory investigation

Article Title: Leptin-producing monocytes in the airway submucosa may contribute to asthma pathogenesis.

doi: 10.1016/j.resinv.2022.09.005

Figure Lengend Snippet: Fig. 4 e Phosphorylation of signaling molecules in response to leptin by asthmatic and Healthy Control (HC) ﬁbroblasts. (A) Normal lung ﬁbroblasts (NHLFs) were seeded in 24-well plates and cultured for 1 h with the following inhibitors of intracellular signaling: AG490 (AG), 100 mM; SB203580 (SB), 10 mM; and U0126 (U), 30 mM. They were then stimulated with 10 mM leptin for 24 h. CCL11/eotaxin and CCL2/MCP-1 levels in the supernatant were determined using Cytometric Bead Array (CBA). Error bars represent the standard error of the mean (SEM) (n ¼ 5e6). *p < 0.05, **p < 0.01. (B) NHLFs were stimulated with 10 mM leptin for 0, 1, 3, 5, 10, or 60 min. The lysates were analyzed by western blotting with anti-p38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, anti-phospho-ERK1/2, or anti-GAPDH antibodies (left panels). Data are representative of 3e4 independent replicates. Quantitative analysis was achieved by densitometry (right panels), and the data are presented as the ratios indicated in the ﬁgure. Error bars represent the SEM (n ¼ 3e4). *p < 0.05, ***p < 0.001 vs.

Article Snippet: The following antibodies were purchased: goat antihuman leptin/OB antibody, mouse anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (IgG1, clone 686613), fluorescein isothiocyanate (FITC)-conjugated mouse anti-human leptin receptor (Ob-R) antibody (IgG2B, clone 52263), and FITC-conjugated mouse isotype control antibody (IgG2A, clone 20102) (R&D Systems, Minneapolis, MN, USA); mouse anti-human CD163 antibody (IgG1, clone 10D6) (Thermo Fisher Scientific, Waltham, MA, USA); and rabbit anti-human phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) antibody, rabbit anti-human p38 MAPK antibody, rabbit anti-human phospho-p44/42 MAPK (extracellular signal-regulated kinase 1/2, ERK1/2) (Thr202/ Tyr204) antibody, and rabbit anti-human p44/42 MAPK (ERK1/ 2) antibody (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Phospho-proteomics, Control, Cell Culture, Western Blot

Expression level of phospho-p38 MAPK. HaCaT cells were cultured with Kumazasa extract (1000 µg/mL) for 1 h, and the protein expression levels of p38 MAPK and phospho-p38 MAPK were analyzed by western blotting (mean ± SD, n = 4, **: p < 0.01 vs. control cells).

Journal: Healthcare

Article Title: Wound-Healing and Skin-Moisturizing Effects of Sasa veitchii Extract

doi: 10.3390/healthcare9060761

Figure Lengend Snippet: Expression level of phospho-p38 MAPK. HaCaT cells were cultured with Kumazasa extract (1000 µg/mL) for 1 h, and the protein expression levels of p38 MAPK and phospho-p38 MAPK were analyzed by western blotting (mean ± SD, n = 4, **: p < 0.01 vs. control cells).

Article Snippet: The membrane was then reacted with the following primary antibodies: rabbit anti-rat AQP3 (Alomone Labs, Jerusalem, Israel), mouse anti-rabbit Na/K-ATPase (Merck Millipore, Darmstadt, Germany), rabbit anti-human phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182) (Cell Signaling Technology, Beverly, MA, USA), and rabbit anti-human p38 MAPK (Cell Signaling Technology) antibodies.

Techniques: Expressing, Cell Culture, Western Blot, Control

UV-B irradiation at 150 mJ/cm 2 induced p38 activation. ( A ) Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then irradiated with UV-B at a level of 150 mJ/cm 2 . The cell/matrix layers were collected at 0, 5, 10, 15, and 30 min after UV irradiation and analyzed by Western blot against p38 and phosphor-p38. The protein levels of p38 did not change, whereas those of phosphor-p38 were maximally detected at 10 min. ( B ) 10 min after irradiation, the cells were labeled for phosphor-p38. Phospho-p38 is labeled in the nucleus at 150 mJ/cm 2 , whereas it is not labeled at 0 mJ/cm 2 . Bar = 50 μm.

Journal: Acta Histochemica et Cytochemica

Article Title: Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules In Vitro

doi: 10.1267/ahc.20-00021

Figure Lengend Snippet: UV-B irradiation at 150 mJ/cm 2 induced p38 activation. ( A ) Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then irradiated with UV-B at a level of 150 mJ/cm 2 . The cell/matrix layers were collected at 0, 5, 10, 15, and 30 min after UV irradiation and analyzed by Western blot against p38 and phosphor-p38. The protein levels of p38 did not change, whereas those of phosphor-p38 were maximally detected at 10 min. ( B ) 10 min after irradiation, the cells were labeled for phosphor-p38. Phospho-p38 is labeled in the nucleus at 150 mJ/cm 2 , whereas it is not labeled at 0 mJ/cm 2 . Bar = 50 μm.

Article Snippet: Then, the cell/matrix layers were incubated for 2 hr with anti-human phospho-p38 mitogen-activated protein kinase (MAPK) polyclonal antibody (#9211: Cell Signaling Technology) and diluted to 1:2000 with blocking buffer for 1 hr at room temperature.

Techniques: Irradiation, Activation Assay, Cell Culture, Western Blot, Labeling

UV-B irradiation increases the level of MMP-2 via p38 activation. Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then the cells pretreated with SB203580 (p38 inhibitor) were irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm 2 . Then, the cells were cultured for another 12 and 24 hr under serum-free culture conditions. ( A ) Cell/matrix layers were collected 10 min after UV irradiation and analyzed by Western blot against phosphor-p38. The level of phosphor-p38 treated with SB203580 was inhibited at the same level as the control (0 mJ/cm 2 ). ( B ) Immunofluorescence showed that phosphor-p38 was labeled in the cells at 150 mJ/cm 2 , but not in SB203580-treated cells. Cell/matrix layers were collected at 12 ( C ) and 24 ( D ) hours after UV irradiation and analyzed by ELISA. The active MMP-2 in the SB203580-treated cells was suppressed compared with the SB203580-untreated vehicle cells. The values for each control sample (0 mJ/cm 2 ) without SB203580 were arbitrarily assigned a value of 1. The results represent the mean ± standard deviation of four independent determinations. ** P < 0.05 versus control.

Journal: Acta Histochemica et Cytochemica

Article Title: Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules In Vitro

doi: 10.1267/ahc.20-00021

Figure Lengend Snippet: UV-B irradiation increases the level of MMP-2 via p38 activation. Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then the cells pretreated with SB203580 (p38 inhibitor) were irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm 2 . Then, the cells were cultured for another 12 and 24 hr under serum-free culture conditions. ( A ) Cell/matrix layers were collected 10 min after UV irradiation and analyzed by Western blot against phosphor-p38. The level of phosphor-p38 treated with SB203580 was inhibited at the same level as the control (0 mJ/cm 2 ). ( B ) Immunofluorescence showed that phosphor-p38 was labeled in the cells at 150 mJ/cm 2 , but not in SB203580-treated cells. Cell/matrix layers were collected at 12 ( C ) and 24 ( D ) hours after UV irradiation and analyzed by ELISA. The active MMP-2 in the SB203580-treated cells was suppressed compared with the SB203580-untreated vehicle cells. The values for each control sample (0 mJ/cm 2 ) without SB203580 were arbitrarily assigned a value of 1. The results represent the mean ± standard deviation of four independent determinations. ** P < 0.05 versus control.

Techniques: Irradiation, Activation Assay, Cell Culture, Western Blot, Control, Immunofluorescence, Labeling, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling

doi: 10.3390/ijms20081826

Figure Lengend Snippet: The effect of p38 activity on FRS2. ( a – c ) FGF1-induced electrophoretic mobility shift of FRS2. Serum-starved ( a , c ) NIH3T3 and ( b ) U2OS-R1 were pretreated for 20 min with or without MEK1/2 inhibitors (20 µM U0126, 1 µM SL327), p38 inhibitor (5 µM SB203580), and p38 activator (10 µM anisomycin), and then stimulated with the growth factor in the presence of heparin (10 U/mL) for 15 min. Cells were lysed, and the cellular material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) ( a , b ) or Phos-Tag SDS-PAGE ( c ) and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 or anti-tubulin as a loading control. ( d ) In vitro phosphorylation of FRS2 by p38α kinase. Recombinant, active kinase p38α and partial recombinant fusion protein FRS2α with GST tag were incubated with (γ- 33 P) ATP in reaction buffer at 30 °C for 30 min in the presence or absence of 5 µM SB203580. Erk1 and Erk2 kinases served as positive controls. The proteins were analyzed by SDS-PAGE, electroblotting, and autoradiography (upper panel), and then the membrane was stained with Coomassie (lower panel). Representative experiments are shown, for ( a ) and ( b ) n = 4, and for ( c ) and ( d ) n = 2.

Article Snippet: GST-tagged FRS2 recombinant protein (Q01) was purchased from Anova (Taipei, Taiwan), active human recombinant p38α MAPK was from R&D Systems (Minneapolis, MN, USA) and active human recombinant MAP kinases, Erk1 (p44) and Erk2 (p42), from Calbiochem.

Techniques: Activity Assay, Electrophoretic Mobility Shift Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Control, In Vitro, Phospho-proteomics, Recombinant, Incubation, Autoradiography, Membrane, Staining

Journal: International Journal of Molecular Sciences

Article Title: Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling

doi: 10.3390/ijms20081826

Figure Lengend Snippet: The crosstalk between p38 and Erk1/2 in downregulation of FGF1-induced signaling. Serum-starved ( a ) NIH3T3 and ( b ) U2OS-R1 cells were pretreated for 30 min with or without 20 µM U0126, 5 µM SB203580 and 10 µM anisomycin, and then stimulated with the growth factor in the presence of heparin (10 U/mL) and brefeldin A (2 μg/mL) for different time points. Cells were lysed, and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-FGFR (Tyr653/Tyr654) (pY-FGFR), anti-phospho-FGFR1 (Ser777) (pS777-FGFR1), anti-phospho-FRS2 (Tyr196) (pY-FRS), anti-FRS2, and anti-Hsp90 as a loading control. Anti-phospho-Erk1/2 (p-Erk1/2) and anti-phospho-p38 MAPK (Thr180/Tyr182) (p-p38) were used to control the effect of U0126 (MEK inhibitor) and anisomycin (p38 activator). Representative experiments are shown, n = 3. The graphs present quantification of bands from panel b corresponding to phospho-FGFR1 (Tyr653/Tyr654) and phospho-FRS2 (Tyr196) normalized to loading control (Hsp90) and expressed as a fraction of the maximal response in the absence of inhibitor. Data are means ± SD of three independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.

Techniques: SDS Page, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling

doi: 10.3390/ijms20081826

Figure Lengend Snippet: The effect of p38 kinase activity on Erk1/2 activity and FGF1-induced signaling. ( a ) The effect of p38 specific inhibitor on Erk1/2 activity. Serum-starved NIH3T3 cells were treated with increasing concentration of the specific p38 kinase inhibitor SB203580 for 30 min. Then, the cells were lysed and the cellular material was analyzed by SDS-PAGE and immunoblotting using the following antibodies: anti-phospho-Erk1/2 (p-Erk1/2), anti-Erk1/2, and anti-Hsp90 antibodies as a loading control. A representative experiment is shown, n = 4. The graph presents quantification of bands corresponding to phospho-Erk1/2 (p-Erk1/2) normalized to loading control (Hsp90) and expressed as a fold of change in comparison with untreated control. Data are means ± SD of four independent experiments; ** p < 0.01, *** p < 0.001. ( b ) Schematic representation of synergistic effect of p38 and Erk1/2 in the downregulation of FGF1-induced signaling through FRS2. FGF1-induced tyrosine phosphorylation of FGFR1 leads to the activation of FRS2 followed by GRB2/SOS-mediated activation of RAS and MAP kinases (Erk1/2 and p38). Activated Erks are supported by p38 in phosphorylation of FRS2 (red arrows), constituting a negative feedback loop that results in reduced tyrosine phosphorylation of FRS2 and consequent attenuation of FGFR signaling. Grey dashed line represents functional cross-talk between Erks and p38.

Techniques: Activity Assay, Concentration Assay, SDS Page, Western Blot, Control, Comparison, Phospho-proteomics, Activation Assay, Functional Assay

Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK

Journal: Arthritis Research & Therapy

Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Intracellular signaling pathways in fibroblasts stimulated with SSc-ICs or NHS-ICs. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK

Article Snippet: Membranes were blocked for 2 h at room temperature in PBS/0.05% Tween 20 (PT) (Bio-Rad Laboratories, Hercules, CA, USA) containing 5% nonfat milk powder (Mellin, Milan, Italy), and incubated with anti-human nuclear factor kappa B (NFκB), anti-human phosphorylated NFκB (pNFκB), anti-human p38 mitogen activated kinase (p38MAPK), anti-human phosphorylated p38MAPK (pp38MAPK), anti-human SAPK-JNK or anti-human phosphorylated SAPK-JNK (anti-pSAPK-JNK) antibodies (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Protein-Protein interactions, Control, Software, Western Blot

Intracellular signaling pathways in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to SLE-ICs, PAPS-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK, PAPS primary anti-phospholipid syndrome, SLE systemic lupus erythematosus

Journal: Arthritis Research & Therapy

Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Intracellular signaling pathways in fibroblasts stimulated with SLE-ICs, PAPS-ICs or NHS-ICs. Fibroblasts exposed to SLE-ICs, PAPS-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. a pNFκB/NFκB; b pp38MAPK/p38MAPK; c pp54SAPK-JNK/p54SAPK-JNK; d pp46SAPK-JNK/p46SAPK-JNK. Results expressed as ratio of phosphorylated to nonphosphorylated forms, evaluated using ImageJ software. Western blot images representative of single experiment. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. IC immune complex, LPS lipopolysaccharide, MAPK mitogen activated kinase, NHS normal healthy subjects, NFκB nuclear factor kappa B, pNFκB phosphorylated NFκB, pp38MAPK phosphorylated p38MAPK, pp54SAPK-JNK phosphorylated p54SAPK-JNK, pp46SAPK-JNK phosphorylated p46SAPK-JNK, PAPS primary anti-phospholipid syndrome, SLE systemic lupus erythematosus

Techniques: Protein-Protein interactions, Control, Software, Western Blot

Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p < 0.01, ** p < 0.001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, NFκB nuclear factor kappa B, NHS normal healthy subjects, MAPK mitogen activated kinase, pp38MAPK phosphorylated p38MAPK

Journal: Arthritis Research & Therapy

Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: Confirmation of efficacy of NFκB and p38MAPK inhibitors by western blot analysis. Cells preincubated for 1 h at 37 °C with inhibitors of NFκB and p38MAPK. Fibroblasts exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) used as control. Results expressed as percentage of inhibition of activated ( a ) NFκB and ( b ) p38MAPK (expressed as ratio of phosphorylated to nonphosphorylated forms). * p < 0.01, ** p < 0.001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, LPS lipopolysaccharide, NFκB nuclear factor kappa B, NHS normal healthy subjects, MAPK mitogen activated kinase, pp38MAPK phosphorylated p38MAPK

Techniques: Western Blot, Control, Inhibition

TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, NHS normal healthy subjects, MAPK p38 mitogen activated kinase, TGF tumor growth factor

Journal: Arthritis Research & Therapy

Article Title: Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts

doi: 10.1186/s13075-018-1689-6

Figure Lengend Snippet: TGF-β1, Pro-collagenIα1, IL-8 and IL-6 in fibroblasts pretreated with p38MAPK inhibitor and incubated with SSc-ICs or NHS-ICs. Fibroblasts pretreated with SB202190 (20 μmol), a p38MAPK inhibitor, and then exposed to SSc-ICs or NHS-ICs (1:2 dilution). LPS (1 μg/ml) and TGF-β1 (10 ng/ml) used as positive controls. Results expressed as percentage of inhibition of a TGF-β1, b Pro-CollagenIα1, c IL-8 and d IL-6 in untreated versus SB202190-treated cells. * p < 0.01, ** p < 0.001, *** p < 0.0001 versus medium. ACA anti-centromeric protein antibodies, anti-Th/To anti-Th/To antibodies, ARA anti-RNA polymerase III antibodies, ATA anti-DNA topoisomerase I antibodies, IC immune complex, IL interleukin, LPS lipopolysaccharide, NHS normal healthy subjects, MAPK p38 mitogen activated kinase, TGF tumor growth factor

Techniques: Incubation, Inhibition

(A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Cells were pretreated with EGF (100ng/ml) and harvested after the indicated times. Lysates were collected, and p38 MAPK phosphorylation levels were analyzed by Western blotting using the antiphospho-p38 antibody. The same membrane was striped and re-probed with a different antibody for measuring total-p38 MAPK protein levels. (B) Caki-1 cells were preincubated with 400pM of the secreted form of the Klotho (KL) protein at the indicated times followed by EGF (100ng/ml) stimulation for additional 5 min. Cell lysates were subjected to Western blot analysis as described in (A). Plots indicate means ± S.E.M of three independent experiments.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Membrane

(A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Journal: Oncotarget

Article Title: Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways

doi: 10.18632/oncotarget.25481

Figure Lengend Snippet: (A) Classical in vitro wound healing assay was performed using near-confluent serum-starved Caki-1 cells grown on collagen type 1. Cells were either pretreated with 400pM Klotho (KL) or 1μM p38 MAPK-specific inhibitor SB203580 as positive control, for 60 min followed by EGF (100ng/ml) treatment. Cell culture images shown here were taken at 0 and 24 h. (B) Plots of quantification of the resultant cell motility values were computed by gap surface area measurements for four selected microscopic fields in each assay condition. The degree of migration is expressed as % wound closure compared with zero time point. The results represent means ± S.E.M of three independent experiments. (C) Wound healing assays performed under 3D settings. Images are microphotographs of Caki-1 cells showing hole-closure of “tissue openings” generated with magnetic pattering as described in the materials and method. Cells were pretreated the same way with either Klotho (KL) or SB203580 and stimulated with EGF as described for the classical wound healing assay. (D) Plots of rate closure of holes for Caki-1 cells as a function of EGF exposure in the presence or absence of KL or SB203580.

Article Snippet: Rabbit anti-human polyclonal active p38 MAPK antibody and SB203580 were purchased from Promega (Madison, WI).

Techniques: In Vitro, Wound Healing Assay, Positive Control, Cell Culture, Migration, Generated

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1) Product Images from "Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling"

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