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  • 99
    Millipore sb202190
    Reversal of resistance of 40AF to 1,25D-induced differentiation is associated with increased expression of CYP24. HL60-G and 40AF cells were treated for 96 h with 10 nM 1,25D (D3) in combination with potentiators of 1,25D-induced differentiation, <t>SB202190,</t> and one of three plant-derived antioxidants, carnosic acid (DCS), silibinin (DSS), or curcumin (DCuS). A: FACS analysis of monocytic surface markers indicates a significant increase in CD11b and CD14 expression on HL60-G and 40AF cells treated with these triple combinations. Bars represent mean values + SEM (n = 5). B: The levels of VDR-induced transcription were determined by qRT-PCR of CYP24 mRNA levels and relative fold values were calculated as described in the Materials and Methods section. Treatment with triple combinations significantly enhanced the expression of VDR-target gene, CYP24 in 40AF cells when compared to vehicle-treated samples. Levels of CYP24 mRNA induced by DCS-treatment of HL60-G cells are shown as a positive control. ** signifies P
    Sb202190, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb202190/product/Millipore
    Average 99 stars, based on 2003 article reviews
    Price from $9.99 to $1999.99
    sb202190 - by Bioz Stars, 2020-05
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    95
    Tocris sb202190
    TGFβ-mediated suppression of CD248 via ALK5 is specific. (A, B) MEF were incubated with TGFβ (3 ng/ml) for 48 hrs in the presence or absence of the inhibitor of phosphorylated ERK1/2, U0126 10 μM (A) or phosphorylated p38, <t>SB202190</t> 10 μM (B) . Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression. TGFβ-coupling to either ERK1/2 or to p38 is not involved in its suppressive effects on CD248.
    Sb202190, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb202190/product/Tocris
    Average 95 stars, based on 220 article reviews
    Price from $9.99 to $1999.99
    sb202190 - by Bioz Stars, 2020-05
    95/100 stars
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    93
    Abcam sb202190
    Phosphorylation of Sox9 and up-regulation of Sox9 protein by TGF-β1 is dependent on p38 activity. ( A , B ) ATDC5 cells were pretreated with different concentrations of SB203580 ( A ) and <t>SB202190</t> ( B ). After 24 hours, cells were treated with vehicle control (−) or TGF-β1 (+) for 24 hours. Cell lysates were used in immunblots to measure p-MAPK-APK2 protein levels. N = 3. ( C ) Cells were pretreated with 10 μM SB203580 (p38i 1), 5 μM SB202190 (p38i 2), or DMSO control for 24 hours, and TGF-β1 (+) or vehicle control (−) was added to the cells, which were incubated for an additional 2 hours. Cell lysates were used in immunoblots to detect p-Sox9 protein, N = 4. ( D ) Cell lysates were also collected 6 hours after treatment with TGF-β1 (+) or vehicle control (−) and used to determine Sox9 protein levels, N = 6. ( E ) Sox9 mRNA levels were measured in cells that were pretreated with p38i 1, p38i 2, or DMSO and then treated with TGF-β1 or vehicle for 6 hours, N = 5. ( F ) ATDC5 cells were transfected with 30 pmol of p38α and p38β siRNA or N.S. siRNA. After 48 hours, cells were treated with TGF-β1 for 2 or 6 hours. Protein lysates were collected and p-Sox9 and Sox9 protein levels were determined by immunoblot, N = 3. ( G ) Papss2 mRNA levels were measured by QPCR after pretreatment with DMSO or p38i followed by treatment with TGF-β1 or vehicle. mRNA levels were determined relative to DMSO/vehicle treated samples. PPIA was used for gene expression normalization. All QPCR data was analyzed using REST software. *** p > 0.001, N = 4. Western blots were cropped for clarity. Examples of uncropped blots are found in Supplementary Figure S4 .
    Sb202190, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb202190/product/Abcam
    Average 93 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    sb202190 - by Bioz Stars, 2020-05
    93/100 stars
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    Image Search Results


    Reversal of resistance of 40AF to 1,25D-induced differentiation is associated with increased expression of CYP24. HL60-G and 40AF cells were treated for 96 h with 10 nM 1,25D (D3) in combination with potentiators of 1,25D-induced differentiation, SB202190, and one of three plant-derived antioxidants, carnosic acid (DCS), silibinin (DSS), or curcumin (DCuS). A: FACS analysis of monocytic surface markers indicates a significant increase in CD11b and CD14 expression on HL60-G and 40AF cells treated with these triple combinations. Bars represent mean values + SEM (n = 5). B: The levels of VDR-induced transcription were determined by qRT-PCR of CYP24 mRNA levels and relative fold values were calculated as described in the Materials and Methods section. Treatment with triple combinations significantly enhanced the expression of VDR-target gene, CYP24 in 40AF cells when compared to vehicle-treated samples. Levels of CYP24 mRNA induced by DCS-treatment of HL60-G cells are shown as a positive control. ** signifies P

    Journal: Journal of cellular physiology

    Article Title: Resistance to 1,25D-Induced Differentiation in Human Acute Myeloid Leukemia HL60-40AF Cells Is Associated With Reduced Transcriptional Activity and Nuclear Localization of the Vitamin D Receptor

    doi: 10.1002/jcp.21150

    Figure Lengend Snippet: Reversal of resistance of 40AF to 1,25D-induced differentiation is associated with increased expression of CYP24. HL60-G and 40AF cells were treated for 96 h with 10 nM 1,25D (D3) in combination with potentiators of 1,25D-induced differentiation, SB202190, and one of three plant-derived antioxidants, carnosic acid (DCS), silibinin (DSS), or curcumin (DCuS). A: FACS analysis of monocytic surface markers indicates a significant increase in CD11b and CD14 expression on HL60-G and 40AF cells treated with these triple combinations. Bars represent mean values + SEM (n = 5). B: The levels of VDR-induced transcription were determined by qRT-PCR of CYP24 mRNA levels and relative fold values were calculated as described in the Materials and Methods section. Treatment with triple combinations significantly enhanced the expression of VDR-target gene, CYP24 in 40AF cells when compared to vehicle-treated samples. Levels of CYP24 mRNA induced by DCS-treatment of HL60-G cells are shown as a positive control. ** signifies P

    Article Snippet: Inhibitor of p38 MAPK, SB202190, was obtained from Calbiochem (La Jolla, CA), carnosic acid from Alexis Pharmaceuticals (Lausen, Switzerland), silibinin and curcumin from Sigma–Aldrich (St. Louis, MO).

    Techniques: Expressing, Derivative Assay, FACS, Quantitative RT-PCR, Positive Control

    Hyperactivation of p38 MAPK is involved in psoriasis-like skin inflammation. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the epidermis of imiquimod-treated (IMQ (+)) and untreated (IMQ (−)) mice. β -actin was used as a loading control. Results are representative of four animals per group. ( b ) Skin from IMQ (−) and IMQ (+) mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of four animals per group. Stained sections are assessed in a blinded fashion by two independent observers. ( c ) Skin from non-psoriasis volunteers (control) and patients with psoriasis (psoriasis) were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of five non-psoriasis volunteers and six patients with psoriasis. Stained sections were assessed in a blinded fashion by two independent observers. ( d ) S100A8, IL-17A, IL-23p19, and interferon γ (IFN γ ) mRNA expression in dorsal skin of imiquimod-treated mice treated with either vehicle or SB202190 was determined by real-time RT-PCR. All values were normalized to GAPDH. Results are displayed as arbitrary units (expression in vehicle-treated skin (vehicle)=1). Data are represented as the mean±S.E.M. ( N =6 in each group). ( e ) Time course of ear swelling in mice treated with either vehicle (dotted line) or SB202190 (solid line) after imiquimod treatment. Ear swelling was measured at the indicated times. Data are mean±S.E.M. ( N =6 in each group). ( f ) Hypothetical model of the PLC δ 1 regulatory mechanisms for barrier integrity. In the keratinocytes of the SG, PIP 2 is hydrolyzed to IP 3 by PLC δ 1, leading to Ca 2+ release from intracellular Ca 2+ stores and [Ca 2+ ] i elevation. [Ca 2+ ] i elevation induces dephosphorylation of NFAT, leading to its nuclear translocation and induction of NFAT target genes. NFAT activation prevents aberrant activation of p38 MAPK through by unknown mechanisms. Thus, RhoA is thus activated, leading to the formation of TJ. Upon proper TJ formation, a normal SC is also formed, which maintains barrier integrity. Without sufficient PLC δ 1 (right panel), PIP 2 is not hydrolyzed and IP 3 -mediated Ca 2+ release and subsequent NFAT activation are inhibited. Without active NFAT, p38 MAPK is hyperactivated, leading to RhoA inhibition and defective TJ and SC formation. This p38 MAPK hyperactivation-mediated barrier defects contribute to the pathogenesis of psoriasis. Statistical significance was assessed using Welch's t -test. * P

    Journal: Cell Death and Differentiation

    Article Title: Phospholipase Cδ1 regulates p38 MAPK activity and skin barrier integrity

    doi: 10.1038/cdd.2017.56

    Figure Lengend Snippet: Hyperactivation of p38 MAPK is involved in psoriasis-like skin inflammation. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the epidermis of imiquimod-treated (IMQ (+)) and untreated (IMQ (−)) mice. β -actin was used as a loading control. Results are representative of four animals per group. ( b ) Skin from IMQ (−) and IMQ (+) mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of four animals per group. Stained sections are assessed in a blinded fashion by two independent observers. ( c ) Skin from non-psoriasis volunteers (control) and patients with psoriasis (psoriasis) were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of five non-psoriasis volunteers and six patients with psoriasis. Stained sections were assessed in a blinded fashion by two independent observers. ( d ) S100A8, IL-17A, IL-23p19, and interferon γ (IFN γ ) mRNA expression in dorsal skin of imiquimod-treated mice treated with either vehicle or SB202190 was determined by real-time RT-PCR. All values were normalized to GAPDH. Results are displayed as arbitrary units (expression in vehicle-treated skin (vehicle)=1). Data are represented as the mean±S.E.M. ( N =6 in each group). ( e ) Time course of ear swelling in mice treated with either vehicle (dotted line) or SB202190 (solid line) after imiquimod treatment. Ear swelling was measured at the indicated times. Data are mean±S.E.M. ( N =6 in each group). ( f ) Hypothetical model of the PLC δ 1 regulatory mechanisms for barrier integrity. In the keratinocytes of the SG, PIP 2 is hydrolyzed to IP 3 by PLC δ 1, leading to Ca 2+ release from intracellular Ca 2+ stores and [Ca 2+ ] i elevation. [Ca 2+ ] i elevation induces dephosphorylation of NFAT, leading to its nuclear translocation and induction of NFAT target genes. NFAT activation prevents aberrant activation of p38 MAPK through by unknown mechanisms. Thus, RhoA is thus activated, leading to the formation of TJ. Upon proper TJ formation, a normal SC is also formed, which maintains barrier integrity. Without sufficient PLC δ 1 (right panel), PIP 2 is not hydrolyzed and IP 3 -mediated Ca 2+ release and subsequent NFAT activation are inhibited. Without active NFAT, p38 MAPK is hyperactivated, leading to RhoA inhibition and defective TJ and SC formation. This p38 MAPK hyperactivation-mediated barrier defects contribute to the pathogenesis of psoriasis. Statistical significance was assessed using Welch's t -test. * P

    Article Snippet: Either 20 μ M SB202190 or 11 R-VIVIT (25 μ M; Millipore) was added to the assay medium for the last 48 or 72 h of culture.

    Techniques: Mouse Assay, Staining, Expressing, Quantitative RT-PCR, Planar Chromatography, De-Phosphorylation Assay, Translocation Assay, Activation Assay, Inhibition

    PLCδ1 silencing activates p38 MAPK. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and grown in medium either with (high Ca 2+ ) or without (low Ca 2+ ) 1.2 mM CaCl 2 . β -actin was used as a loading control. Results are representative of two trials. ( b ) Extracellular ATP concentrations were measured in conditioned medium of NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs. ( N =4 in each group). ( c ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the presence of an NFAT inhibitor (11 R-VIVIT; treated for 24 h with the concentrations indicated). β -actin was used as a loading control. Results are representative of two trials. ( d , e ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs ( d ) and the epidermis of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice ( e ). β -actin was used as a loading control. Results are representative of two trials ( d ) or three animals per group ( e ). ( f ) Skin from control and PLC δ 1 cKO mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of three animals per group. Stained sections were assessed in a blinded fashion by two independent observers. ( g ) Immunoblotting of phosphorylated (p-HSP27) and total HSP27 in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and either vehicle or a p38 MAPK inhibitor (SB202190). β -actin was used as a loading control. Results are representative of two trials. ( h ) NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs were pretreated with either vehicle or SB202190, and then exposed to 2 mM CaCl 2 for 15 min. RhoA-GTP levels were determined using G-LISA in NHEK stimulated with Ca 2+ . RhoA-GTP levels were normalized to the total protein amount. Data are represented as mean±S.E.M. ( N =3 in each group). ( i ) Immunofluorescence detection of ZO-1 in NHEK treated with either non- (control) or PLCδ1 -targeting siRNAs, grown in medium containing 1.2 mM CaCl 2 for 24 h in the presence of either vehicle or SB202190. Scale bar=50 μ m. Images are representative of three trials. ( j ) Skin samples of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice treated with either vehicle or SB202190, and were stained with H E or an antibody against ZO-1. Dotted lines denote the skin surface. Scale bar=50 μ m (H E), and 20 μ m (ZO-1). Images are representative of five animals per group. ( k ) Lucifer yellow fluorescence was visualized in sections of human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (PLC δ 1siRNA) siRNAs. Either vehicle or SB202190 was added to the medium in the last 72 h of culture. Nuclei were counter-stained with Hoechst (blue). Dotted lines denote the skin culture surface. Scale bar=30 μ m. Images are representative of three trials. Stained sections were assessed in a blinded fashion by two independent observers. ( a , c , d , e and g ) Relative ratios of phosphorylated to total protein (Phospho/Total) are denoted below each panel. Statistical significance was assessed using Welch's t -test. n.s; not significant

    Journal: Cell Death and Differentiation

    Article Title: Phospholipase Cδ1 regulates p38 MAPK activity and skin barrier integrity

    doi: 10.1038/cdd.2017.56

    Figure Lengend Snippet: PLCδ1 silencing activates p38 MAPK. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and grown in medium either with (high Ca 2+ ) or without (low Ca 2+ ) 1.2 mM CaCl 2 . β -actin was used as a loading control. Results are representative of two trials. ( b ) Extracellular ATP concentrations were measured in conditioned medium of NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs. ( N =4 in each group). ( c ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the presence of an NFAT inhibitor (11 R-VIVIT; treated for 24 h with the concentrations indicated). β -actin was used as a loading control. Results are representative of two trials. ( d , e ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs ( d ) and the epidermis of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice ( e ). β -actin was used as a loading control. Results are representative of two trials ( d ) or three animals per group ( e ). ( f ) Skin from control and PLC δ 1 cKO mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of three animals per group. Stained sections were assessed in a blinded fashion by two independent observers. ( g ) Immunoblotting of phosphorylated (p-HSP27) and total HSP27 in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and either vehicle or a p38 MAPK inhibitor (SB202190). β -actin was used as a loading control. Results are representative of two trials. ( h ) NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs were pretreated with either vehicle or SB202190, and then exposed to 2 mM CaCl 2 for 15 min. RhoA-GTP levels were determined using G-LISA in NHEK stimulated with Ca 2+ . RhoA-GTP levels were normalized to the total protein amount. Data are represented as mean±S.E.M. ( N =3 in each group). ( i ) Immunofluorescence detection of ZO-1 in NHEK treated with either non- (control) or PLCδ1 -targeting siRNAs, grown in medium containing 1.2 mM CaCl 2 for 24 h in the presence of either vehicle or SB202190. Scale bar=50 μ m. Images are representative of three trials. ( j ) Skin samples of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice treated with either vehicle or SB202190, and were stained with H E or an antibody against ZO-1. Dotted lines denote the skin surface. Scale bar=50 μ m (H E), and 20 μ m (ZO-1). Images are representative of five animals per group. ( k ) Lucifer yellow fluorescence was visualized in sections of human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (PLC δ 1siRNA) siRNAs. Either vehicle or SB202190 was added to the medium in the last 72 h of culture. Nuclei were counter-stained with Hoechst (blue). Dotted lines denote the skin culture surface. Scale bar=30 μ m. Images are representative of three trials. Stained sections were assessed in a blinded fashion by two independent observers. ( a , c , d , e and g ) Relative ratios of phosphorylated to total protein (Phospho/Total) are denoted below each panel. Statistical significance was assessed using Welch's t -test. n.s; not significant

    Article Snippet: Either 20 μ M SB202190 or 11 R-VIVIT (25 μ M; Millipore) was added to the assay medium for the last 48 or 72 h of culture.

    Techniques: Planar Chromatography, Knock-Out, Mouse Assay, Staining, Immunofluorescence, Fluorescence

    NO • stabilizes ( A ) MAP3K7IP2, ( B ) MRPS18A and ( C ) TP53BP2 mRNA through Erk1/2 as determined by RT–PCR. Left panels show the effects of NO • and the p38 MAPK inhibitor, SB202190 (SB; 0.1 µM), on mRNA degradation. Right panels show the effects of the Erk1/2 inhibitor, PD98059 (PD; 30 µM), on mRNA degradation in the presence of SB. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with transcription inhibitor ActD (2.5 µg/ml) in the absence or presence of indicated MAPK inhibitors, cells were incubated with GSNO (400 µM) or GSH control (400 µM) for 0–180 min. All mRNA levels were quantitated by TaqMan ® RT–PCR and normalized to GADPH mRNA. Data, presented as percentage relative to mRNA levels at 0 min, are the mean ± SEM of three independent experiments. The respective mRNA half-lives of MAP3K7IP2, MRPS18A and TP53BP2 were as follows: 179, 98 and 91 min for control GSH; 236, 132 and 121 min for GSNO; 200, 103 and 119 min for SB/GSH; 314, 166 and 155 min for SB/GSNO; 171, 89 and 100 min for SB/PD/GSH; and 160, 90 and 103 min for SB/PD/GSNO.

    Journal: Nucleic Acids Research

    Article Title: Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements

    doi: 10.1093/nar/gkl386

    Figure Lengend Snippet: NO • stabilizes ( A ) MAP3K7IP2, ( B ) MRPS18A and ( C ) TP53BP2 mRNA through Erk1/2 as determined by RT–PCR. Left panels show the effects of NO • and the p38 MAPK inhibitor, SB202190 (SB; 0.1 µM), on mRNA degradation. Right panels show the effects of the Erk1/2 inhibitor, PD98059 (PD; 30 µM), on mRNA degradation in the presence of SB. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with transcription inhibitor ActD (2.5 µg/ml) in the absence or presence of indicated MAPK inhibitors, cells were incubated with GSNO (400 µM) or GSH control (400 µM) for 0–180 min. All mRNA levels were quantitated by TaqMan ® RT–PCR and normalized to GADPH mRNA. Data, presented as percentage relative to mRNA levels at 0 min, are the mean ± SEM of three independent experiments. The respective mRNA half-lives of MAP3K7IP2, MRPS18A and TP53BP2 were as follows: 179, 98 and 91 min for control GSH; 236, 132 and 121 min for GSNO; 200, 103 and 119 min for SB/GSH; 314, 166 and 155 min for SB/GSNO; 171, 89 and 100 min for SB/PD/GSH; and 160, 90 and 103 min for SB/PD/GSNO.

    Article Snippet: S-nitrosoglutathione (GSNO), SB202190 (SB) and PD98059 (PD) were purchased from Calbiochem (San Diego, CA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Incubation

    Role of hnRNP K and hnRNP E2/E1 in NO • -Erk1/2-CURE signaling. ( A ) RNA REMSAs with either a consensus (left panel) or MAP3K7IP2 (right panel) CURE riboprobes. GSNO (400 µM) treatment for 3 h increased complex formation compared to control GSH; anti-hnRNP K and anti-hnRNP E2/E1 both super-shift the complex; the unlabeled CURE riboprobes, but not the mutant of consensus CURE (mutant CURE) compete with the labeled CURE riboprobes. ( B ) Translocation of hnRNP K and hnRNP E2/E1 to the cytoplasm by western blotting. GSNO (400 µM) treatment for 3 h increased the presence of hnRNP K and hnRNP E2/E1 in the cytoplasm but not in whole-cell lysates compared to control GSH. This effect was further enhanced by the p38 MAPK inhibitor SB202190 (SB; 0.1 µM), but blocked by the Erk1/2 inhibitor PD98059 (PD; 30 µM). A control protein α tubulin is shown for comparison. Experiments in (A and B) were repeated at least twice with similar results. ( C ) Overexpression of hnRNP K mimicked the effect of NO • , repressing the expression of a chimeric LUC-CURE reporter gene. THP-1 cells were co-transfected with pGL3/CURE, pGL3/CUREmut or control pGL3 and pcDNA3 (empty vector) or phnRNP-K (hnRNP K expression plasmid). After treatment with GSH (400 µM) or GSNO (400 µM) for 20 h, LUC activities were measured. Data, presented as percentage relative to LUC activity with pGL3, are the mean ± SEM of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements

    doi: 10.1093/nar/gkl386

    Figure Lengend Snippet: Role of hnRNP K and hnRNP E2/E1 in NO • -Erk1/2-CURE signaling. ( A ) RNA REMSAs with either a consensus (left panel) or MAP3K7IP2 (right panel) CURE riboprobes. GSNO (400 µM) treatment for 3 h increased complex formation compared to control GSH; anti-hnRNP K and anti-hnRNP E2/E1 both super-shift the complex; the unlabeled CURE riboprobes, but not the mutant of consensus CURE (mutant CURE) compete with the labeled CURE riboprobes. ( B ) Translocation of hnRNP K and hnRNP E2/E1 to the cytoplasm by western blotting. GSNO (400 µM) treatment for 3 h increased the presence of hnRNP K and hnRNP E2/E1 in the cytoplasm but not in whole-cell lysates compared to control GSH. This effect was further enhanced by the p38 MAPK inhibitor SB202190 (SB; 0.1 µM), but blocked by the Erk1/2 inhibitor PD98059 (PD; 30 µM). A control protein α tubulin is shown for comparison. Experiments in (A and B) were repeated at least twice with similar results. ( C ) Overexpression of hnRNP K mimicked the effect of NO • , repressing the expression of a chimeric LUC-CURE reporter gene. THP-1 cells were co-transfected with pGL3/CURE, pGL3/CUREmut or control pGL3 and pcDNA3 (empty vector) or phnRNP-K (hnRNP K expression plasmid). After treatment with GSH (400 µM) or GSNO (400 µM) for 20 h, LUC activities were measured. Data, presented as percentage relative to LUC activity with pGL3, are the mean ± SEM of three independent experiments.

    Article Snippet: S-nitrosoglutathione (GSNO), SB202190 (SB) and PD98059 (PD) were purchased from Calbiochem (San Diego, CA).

    Techniques: Mutagenesis, Labeling, Translocation Assay, Western Blot, Over Expression, Expressing, Transfection, Plasmid Preparation, Activity Assay

    Heat maps; effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on mRNA degradation as determined by microarray. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with ActD (2.5 µg/ml) in the absence or presence of SB (0.1 µM), cells were incubated with GSNO (400 µM) or control GSH (400 µM) for 0–180 min. At the indicated time-points, cells were harvested to extract total RNA for microarray analysis. The half-lives of 220 genes were found to be differentially regulated (see Materials and Methods). ( A ) Hierarchical clustering of normalized mean signal intensities from four independent experiments for all 220 genes at each time point and condition. ( B ) Same results as (A) after conversion of individual time point data into slopes based on a first order mRNA decay model.

    Journal: Nucleic Acids Research

    Article Title: Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements

    doi: 10.1093/nar/gkl386

    Figure Lengend Snippet: Heat maps; effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on mRNA degradation as determined by microarray. THP-1 cells (2 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with ActD (2.5 µg/ml) in the absence or presence of SB (0.1 µM), cells were incubated with GSNO (400 µM) or control GSH (400 µM) for 0–180 min. At the indicated time-points, cells were harvested to extract total RNA for microarray analysis. The half-lives of 220 genes were found to be differentially regulated (see Materials and Methods). ( A ) Hierarchical clustering of normalized mean signal intensities from four independent experiments for all 220 genes at each time point and condition. ( B ) Same results as (A) after conversion of individual time point data into slopes based on a first order mRNA decay model.

    Article Snippet: S-nitrosoglutathione (GSNO), SB202190 (SB) and PD98059 (PD) were purchased from Calbiochem (San Diego, CA).

    Techniques: Microarray, Incubation

    Effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on MAPK phosphorylation. ( A ) NO • increases p38 MAPK phosphorylation, an effect blocked by SB (0.1 µM). ( B ) NO • increases Erk1/2 phosphorylation, an effect enhanced by SB (0.1 µM). ( C ) SB (0–5 µM) alone increases Erk1/2 phosphorylation. THP-1 cells (1 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with ActD (2.5 µg/ml) in the absence (control) or presence of SB, cells were incubated without or with GSNO (0–800 µM) for another 30 min, as indicated and then lysed. Each experiment was repeated at least twice with similar results.

    Journal: Nucleic Acids Research

    Article Title: Nitric oxide activation of Erk1/2 regulates the stability and translation of mRNA transcripts containing CU-rich elements

    doi: 10.1093/nar/gkl386

    Figure Lengend Snippet: Effects of NO • and the p38 MAPK inhibitor SB202190 (SB) on MAPK phosphorylation. ( A ) NO • increases p38 MAPK phosphorylation, an effect blocked by SB (0.1 µM). ( B ) NO • increases Erk1/2 phosphorylation, an effect enhanced by SB (0.1 µM). ( C ) SB (0–5 µM) alone increases Erk1/2 phosphorylation. THP-1 cells (1 × 10 7 ) were stimulated with LPS (1 µg/ml) for 4 h. After 30 min treatment with ActD (2.5 µg/ml) in the absence (control) or presence of SB, cells were incubated without or with GSNO (0–800 µM) for another 30 min, as indicated and then lysed. Each experiment was repeated at least twice with similar results.

    Article Snippet: S-nitrosoglutathione (GSNO), SB202190 (SB) and PD98059 (PD) were purchased from Calbiochem (San Diego, CA).

    Techniques: Incubation

    The induction of Th17 cells by sodium chloride depends on p38/MAPK, NFAT5 and SGK1 a , Naive CD4 + cells were stimulated in the presence (NaCl) or absence (none) of additional 40 mM NaCl and were analysed by FACS for phosphorylated p38 (p-p38; n = 5). b, Naïve CD4 cells were differentiated into Th17 cells as indicated in the presence or absence of NaCl and SB202190 (p38i) and analysed by qRT-PCR as depicted in the bar graph (n=7) or by FACS (the left row shows cells differentiated in the absence of TGF-β1). c, Naïve CD4 cells were stimulated for 3h in the presence or absence of NaCl and SB202190 and analysed by qRT-PCR for NFAT5 (n=4). d, Cells were transduced with NFAT5 specific (shNFAT5) or control shRNA (control), stimulated like in b) and analysed by FACS. The bar graphs depict qRT-PCR analyses of NFAT5 , IL-17A and SLC5A3 (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shNFAT5, one representative experiment of four is shown). e, Cells were stimulated like in c), but analysed by qRT-PCR for SGK1 (n=4). f, Cells were transduced with a shRNA specific for SGK1 (shSGK1) or a control shRNA (control) and activated like in b), and analysed by FACS. Expression of SGK1 and IL-17A was determined by qRT-PCR (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shSGK1, one representative experiment of four is shown). g, Cells were cultured like in b), but in the presence or absence of the SGK1 inhibitor GSK650394 (SGK1i) and analysed by FACS. The bar graph shows qRT-PCR for IL-17A under similar conditions (n=5). FACS and qRT-PCR (relative expression) data depicted in bar graphs were normalised to controls.

    Journal: Nature

    Article Title: Sodium Chloride Drives Autoimmune Disease by the Induction of Pathogenic Th17 Cells

    doi: 10.1038/nature11868

    Figure Lengend Snippet: The induction of Th17 cells by sodium chloride depends on p38/MAPK, NFAT5 and SGK1 a , Naive CD4 + cells were stimulated in the presence (NaCl) or absence (none) of additional 40 mM NaCl and were analysed by FACS for phosphorylated p38 (p-p38; n = 5). b, Naïve CD4 cells were differentiated into Th17 cells as indicated in the presence or absence of NaCl and SB202190 (p38i) and analysed by qRT-PCR as depicted in the bar graph (n=7) or by FACS (the left row shows cells differentiated in the absence of TGF-β1). c, Naïve CD4 cells were stimulated for 3h in the presence or absence of NaCl and SB202190 and analysed by qRT-PCR for NFAT5 (n=4). d, Cells were transduced with NFAT5 specific (shNFAT5) or control shRNA (control), stimulated like in b) and analysed by FACS. The bar graphs depict qRT-PCR analyses of NFAT5 , IL-17A and SLC5A3 (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shNFAT5, one representative experiment of four is shown). e, Cells were stimulated like in c), but analysed by qRT-PCR for SGK1 (n=4). f, Cells were transduced with a shRNA specific for SGK1 (shSGK1) or a control shRNA (control) and activated like in b), and analysed by FACS. Expression of SGK1 and IL-17A was determined by qRT-PCR (n=5). CCR6 was analysed by FACS (black histogram: control, grey histogram: shSGK1, one representative experiment of four is shown). g, Cells were cultured like in b), but in the presence or absence of the SGK1 inhibitor GSK650394 (SGK1i) and analysed by FACS. The bar graph shows qRT-PCR for IL-17A under similar conditions (n=5). FACS and qRT-PCR (relative expression) data depicted in bar graphs were normalised to controls.

    Article Snippet: In some experiments the specific inhibitors SB202190 (Sigma Aldrich, St Louis, MO) or GSK650394 (Tocris Bioscience/R & D Systems) at concentrations of 5 μM or 1 μM respectively, were added to the cultures.

    Techniques: FACS, Quantitative RT-PCR, Transduction, shRNA, Expressing, Cell Culture

    Impact of HO-1 siRNA on effects of SB202190 on metabolic activity, apoptosis and autophagy HUVEC were transfected with HO-1-specific siRNA or non-targeting siRNA (NON) 24 h prior to stimulation with SB202190 (10 μM). Following a 24-h incubation, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for autophagy-related protein LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 13–16 (A), n = 18–20 (B) or n = 20 (C, D) experiments. * P

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: Impact of HO-1 siRNA on effects of SB202190 on metabolic activity, apoptosis and autophagy HUVEC were transfected with HO-1-specific siRNA or non-targeting siRNA (NON) 24 h prior to stimulation with SB202190 (10 μM). Following a 24-h incubation, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for autophagy-related protein LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 13–16 (A), n = 18–20 (B) or n = 20 (C, D) experiments. * P

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activity Assay, Transfection, Incubation, Expressing

    Proposed mechanism underlying the cytoprotective action of SB202190 in HUVEC The p38 MAPK inhibitor SB202190 inhibits apoptosis of endothelial cells by activation of autophagy followed by induction of the cytoprotective enzyme HO-1. Activation of autophagy was substantiated by increased metabolic activity and enhanced phosphatidyl ethanolamine (PE)-conjugation of LC3-I protein, resulting in LC3-II protein [ 43 ]. Inhibition or knockdown of HO-1 reversed anti-apoptotic effects, but not autophagy activation mediated by SB202190. Inhibition of autophagosome formation by late-phase autophagy inhibitor bafilomycin A 1 [ 44 , 48 ] reversed pro-metabolic effects and HO-1 induction by SB202190 thereby abolishing anti-apoptotic effects of the p38 MAPK inhibitor in HUVEC.

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: Proposed mechanism underlying the cytoprotective action of SB202190 in HUVEC The p38 MAPK inhibitor SB202190 inhibits apoptosis of endothelial cells by activation of autophagy followed by induction of the cytoprotective enzyme HO-1. Activation of autophagy was substantiated by increased metabolic activity and enhanced phosphatidyl ethanolamine (PE)-conjugation of LC3-I protein, resulting in LC3-II protein [ 43 ]. Inhibition or knockdown of HO-1 reversed anti-apoptotic effects, but not autophagy activation mediated by SB202190. Inhibition of autophagosome formation by late-phase autophagy inhibitor bafilomycin A 1 [ 44 , 48 ] reversed pro-metabolic effects and HO-1 induction by SB202190 thereby abolishing anti-apoptotic effects of the p38 MAPK inhibitor in HUVEC.

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activation Assay, Activity Assay, Conjugation Assay, Inhibition

    Impact of SB202190 on metabolic activity, apoptosis and cell cycle progression of HUVEC Cells were incubated with increasing concentrations of SB202190 or vehicle control for 24 h (A, C, E) or with 10 μM SB202190 for the indicated times (B, D) . Following incubation, cells were analysed for metabolic activity using WST-1 colorimetric assay (A, B), DNA fragmentation (C, D) or cell cycle distribution using flow cytometry (E). Exemplary images of flow cytometry analysis are shown for vehicle control and 10 μM SB202190, respectively (F) . Percent control represents comparison with the respective vehicle-treated time-matched group. Values are means ± SEM of n = 15–18 (A), n = 19–22 (B), n = 7–8 (C), n = 11–12 (D) and n = 3 (E) experiments. * P

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: Impact of SB202190 on metabolic activity, apoptosis and cell cycle progression of HUVEC Cells were incubated with increasing concentrations of SB202190 or vehicle control for 24 h (A, C, E) or with 10 μM SB202190 for the indicated times (B, D) . Following incubation, cells were analysed for metabolic activity using WST-1 colorimetric assay (A, B), DNA fragmentation (C, D) or cell cycle distribution using flow cytometry (E). Exemplary images of flow cytometry analysis are shown for vehicle control and 10 μM SB202190, respectively (F) . Percent control represents comparison with the respective vehicle-treated time-matched group. Values are means ± SEM of n = 15–18 (A), n = 19–22 (B), n = 7–8 (C), n = 11–12 (D) and n = 3 (E) experiments. * P

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activity Assay, Incubation, Colorimetric Assay, Flow Cytometry, Cytometry

    SB202190 triggers activation of autophagy in HUVEC Cells were incubated with increasing concentrations of SB202190 or vehicle control for 24 h (A) or with 10 μM SB202190 for the indicated times (B) . Following incubation, cells were harvested and lysates were analysed for autophagy-related protein LC3A/B-I/II. Protein expression values were normalised to β-actin. Percent control represents comparison with the respective vehicle-treated time-matched group (set as 100%). Values are means ± SEM of n = 4 (A) or n = 3 (B) experiments. Statistical analysis revealed no significant differences between SB202190-treated groups and time-matched vehicle controls.

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: SB202190 triggers activation of autophagy in HUVEC Cells were incubated with increasing concentrations of SB202190 or vehicle control for 24 h (A) or with 10 μM SB202190 for the indicated times (B) . Following incubation, cells were harvested and lysates were analysed for autophagy-related protein LC3A/B-I/II. Protein expression values were normalised to β-actin. Percent control represents comparison with the respective vehicle-treated time-matched group (set as 100%). Values are means ± SEM of n = 4 (A) or n = 3 (B) experiments. Statistical analysis revealed no significant differences between SB202190-treated groups and time-matched vehicle controls.

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activation Assay, Incubation, Expressing

    Impact of the HO-1 inhibitor SnPPIX on effects of SB202190 on metabolic activity, apoptosis and autophagy HUVEC were pre-incubated with the HO-1 activity inhibitor SnPPIX (25 μM) for 1 h followed by addition of SB202190 (10 μM) and continuation of incubation for another 24 h. Thereafter, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for autophagy-related protein LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 21–24 (A), n = 10–12 (B), n = 12 (C) or n = 16 (D) experiments. * P

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: Impact of the HO-1 inhibitor SnPPIX on effects of SB202190 on metabolic activity, apoptosis and autophagy HUVEC were pre-incubated with the HO-1 activity inhibitor SnPPIX (25 μM) for 1 h followed by addition of SB202190 (10 μM) and continuation of incubation for another 24 h. Thereafter, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for autophagy-related protein LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 21–24 (A), n = 10–12 (B), n = 12 (C) or n = 16 (D) experiments. * P

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activity Assay, Incubation, Expressing

    Impact of the autophagy inhibitor bafilomycin A 1 on effects of SB202190 on metabolic activity, apoptosis, autophagy and HO-1 expression HUVEC were pre-incubated with the autophagy inhibitor bafilomycin A 1 (2.5 nM) for 1 h followed by addition of SB202190 (10 μM) and continuation of incubation for another 24 h. Thereafter, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for protein expression of LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 19–25 (A), n = 14–15 (B), n = 11 (C) and n = 12 (D) experiments. * P

    Journal: Oncotarget

    Article Title: SB202190 inhibits endothelial cell apoptosis via induction of autophagy and heme oxygenase-1

    doi: 10.18632/oncotarget.25234

    Figure Lengend Snippet: Impact of the autophagy inhibitor bafilomycin A 1 on effects of SB202190 on metabolic activity, apoptosis, autophagy and HO-1 expression HUVEC were pre-incubated with the autophagy inhibitor bafilomycin A 1 (2.5 nM) for 1 h followed by addition of SB202190 (10 μM) and continuation of incubation for another 24 h. Thereafter, cells were analysed for metabolic activity (A) and DNA fragmentation (B) . Lysates of cells were analysed for protein expression of LC3A/B-I/II (C) and HO-1 (D) . Protein expression values were normalised to β-actin. Percent control represents comparison with vehicle-treated group (set as 100%). Values are means ± SEM of n = 19–25 (A), n = 14–15 (B), n = 11 (C) and n = 12 (D) experiments. * P

    Article Snippet: Materials SB202190 was obtained from Sigma-Aldrich (Taufkirchen, Germany).

    Techniques: Activity Assay, Expressing, Incubation

    Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.

    Journal: PLoS ONE

    Article Title: Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase, and Promotes the Degradation of Cdt1 following UV Irradiation

    doi: 10.1371/journal.pone.0046480

    Figure Lengend Snippet: Cdt2 phosphorylation after UV irradiation is caffeine- and MAP kinase-inhibitor sensitive. A. HeLa cells synchronized in the G1 phase were treated with the indicated inhibitors for 2 h and UV-irradiated (+) or not (−). Cell extract was blotted with Cdt1 and Cdt2 antibodies. Inhibitors used were: ERKi, U0126; JNKi, SP600125; p38i, SB202190. The asterisk indicates a non-specific band, and used as a loading control. B. Asynchronously growing HeLa cells were treated with the indicated MAP kinase inhibitors and UV-irradiated (+) or not. One hour later, cells were examined for Cdt2 phosphorylation levels and Cdt1 protein levels. Cdt2 phosphorylation levels were examined using Phos-tag gel. C. Target specificity of inhibitors. Activation of each MAP kinase was examined using specific phosphopeptide antibodies (Erk1 and 2, JNK or p38) before (−) and after (+) UV-irradiation (C-1 and 2). Cells, treated or not with the indicated inhibitors for 2 h, were UV-irradiated or not. One hour later, cell extracts were prepared and their phosphorylation levels of ERK (Erk1 and Erk2), Jun, and MAPKAPK2 (MK2) were examined(C-2). Note that U0126 inhibits the ERK pathway by inhibiting ERK1-activating kinases MEK1 and MEK2. Jun is a JNK substrate and MAPKAPK2 is a p38 substrate. Arrow in MK2 Western blotting indicates the phosphorylated form of MK2. The asterisk indicates a non-specific band.

    Article Snippet: MAP kinase inhibitors were used at the following concentrations: ERK inhibitor U0126 (Calbiochem) at 10 µM, JNK inhibitor II (SP600125, Calbiochem) at 30 µM and p38 inhibitor SB202190 (Calbiochem) at 20 µM.

    Techniques: Irradiation, Activation Assay, Western Blot

    Inhibition of p38MAPK blocks PV IgG-induced DSG3 internalization (18-h time point). Keratinocytes were treated with PBS, NH IgG (2 mg/ml), or PV IgG (2 mg/ml) for 18 h ( A ) or first pretreated with the p38MAPK inhibitor SB202190 (+ INH ) followed by PBS,

    Journal: The Journal of Biological Chemistry

    Article Title: p38MAPK Signaling and Desmoglein-3 Internalization Are Linked Events in Pemphigus Acantholysis *

    doi: 10.1074/jbc.M109.087999

    Figure Lengend Snippet: Inhibition of p38MAPK blocks PV IgG-induced DSG3 internalization (18-h time point). Keratinocytes were treated with PBS, NH IgG (2 mg/ml), or PV IgG (2 mg/ml) for 18 h ( A ) or first pretreated with the p38MAPK inhibitor SB202190 (+ INH ) followed by PBS,

    Article Snippet: Two hours prior to treating cells, keratinocytes were preincubated with the p38MAPK inhibitor SB202190 (100 μ m ) or Me2 SO vehicle control at 37 °C.

    Techniques: Inhibition

    Inhibition of p38MAPK blocks PV IgG-induced DSG3 internalization (4-h time point). Keratinocytes were treated with PBS, NH IgG (2 mg/ml), or PV IgG (2 mg/ml) for 4 h ( A ) or first pretreated with the p38MAPK inhibitor SB202190 (+ INH ) followed by PBS, NH

    Journal: The Journal of Biological Chemistry

    Article Title: p38MAPK Signaling and Desmoglein-3 Internalization Are Linked Events in Pemphigus Acantholysis *

    doi: 10.1074/jbc.M109.087999

    Figure Lengend Snippet: Inhibition of p38MAPK blocks PV IgG-induced DSG3 internalization (4-h time point). Keratinocytes were treated with PBS, NH IgG (2 mg/ml), or PV IgG (2 mg/ml) for 4 h ( A ) or first pretreated with the p38MAPK inhibitor SB202190 (+ INH ) followed by PBS, NH

    Article Snippet: Two hours prior to treating cells, keratinocytes were preincubated with the p38MAPK inhibitor SB202190 (100 μ m ) or Me2 SO vehicle control at 37 °C.

    Techniques: Inhibition

    PV IgG-induced depletion of DSG3 is blocked by the p38MAPK inhibitor SB202190. Primary human keratinocytes were pretreated for 1 h with 100 μ m SB202190 or vehicle and then treated with PBS ( Con ), control IgG ( Con IgG ; 2 mg/ml), or PV IgG (2 mg/ml)

    Journal: The Journal of Biological Chemistry

    Article Title: p38MAPK Signaling and Desmoglein-3 Internalization Are Linked Events in Pemphigus Acantholysis *

    doi: 10.1074/jbc.M109.087999

    Figure Lengend Snippet: PV IgG-induced depletion of DSG3 is blocked by the p38MAPK inhibitor SB202190. Primary human keratinocytes were pretreated for 1 h with 100 μ m SB202190 or vehicle and then treated with PBS ( Con ), control IgG ( Con IgG ; 2 mg/ml), or PV IgG (2 mg/ml)

    Article Snippet: Two hours prior to treating cells, keratinocytes were preincubated with the p38MAPK inhibitor SB202190 (100 μ m ) or Me2 SO vehicle control at 37 °C.

    Techniques:

    Pharmacologic inhibition of p38α/β blocks TMZ-induced p38 activity, G 2 arrest, and activation of G 2 checkpoint proteins. U87 human glioma cells were pretreated with SB202190 or SB203580 (20 μM, 1 h), after which the cells were incubated with TMZ (3 h, 100 μM) in the presence of SB compounds, washed, and plated in SB compound-containing medium. Cells were then either fractionated and assayed for levels of phosphorylation of the p38 substrate MAPKAP2 (A), analyzed for extent of G 2 arrest by FACS analysis (B), or analyzed for nuclear activation of the G 2 checkpoint proteins Chk1, Chk2, Cdc25C, or Cdc2 by Western blotting using phospho-specific antibodies (C). In panel C, the mean fold induction of protein levels based on densitometric analysis is displayed below each immunoreactive band. Histone H1 and tubulin were used as loading controls and as controls for cell fractionation. The data shown are representative of three experiments. *, values differing statistically ( P

    Journal: Molecular and Cellular Biology

    Article Title: The p38 Mitogen-Activated Protein Kinase Pathway Links the DNA Mismatch Repair System to the G2 Checkpoint and to Resistance to Chemotherapeutic DNA-Methylating Agents

    doi: 10.1128/MCB.23.22.8306-8315.2003

    Figure Lengend Snippet: Pharmacologic inhibition of p38α/β blocks TMZ-induced p38 activity, G 2 arrest, and activation of G 2 checkpoint proteins. U87 human glioma cells were pretreated with SB202190 or SB203580 (20 μM, 1 h), after which the cells were incubated with TMZ (3 h, 100 μM) in the presence of SB compounds, washed, and plated in SB compound-containing medium. Cells were then either fractionated and assayed for levels of phosphorylation of the p38 substrate MAPKAP2 (A), analyzed for extent of G 2 arrest by FACS analysis (B), or analyzed for nuclear activation of the G 2 checkpoint proteins Chk1, Chk2, Cdc25C, or Cdc2 by Western blotting using phospho-specific antibodies (C). In panel C, the mean fold induction of protein levels based on densitometric analysis is displayed below each immunoreactive band. Histone H1 and tubulin were used as loading controls and as controls for cell fractionation. The data shown are representative of three experiments. *, values differing statistically ( P

    Article Snippet: The p38 inhibitors SB202190 and SB203580 were purchased from Calbiochem (San Diego, Calif.) and dissolved in DMSO.

    Techniques: Inhibition, Activity Assay, Activation Assay, Incubation, FACS, Western Blot, Cell Fractionation

    TGFβ-mediated suppression of CD248 via ALK5 is specific. (A, B) MEF were incubated with TGFβ (3 ng/ml) for 48 hrs in the presence or absence of the inhibitor of phosphorylated ERK1/2, U0126 10 μM (A) or phosphorylated p38, SB202190 10 μM (B) . Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression. TGFβ-coupling to either ERK1/2 or to p38 is not involved in its suppressive effects on CD248.

    Journal: BMC Cancer

    Article Title: TGF?-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells

    doi: 10.1186/1471-2407-14-113

    Figure Lengend Snippet: TGFβ-mediated suppression of CD248 via ALK5 is specific. (A, B) MEF were incubated with TGFβ (3 ng/ml) for 48 hrs in the presence or absence of the inhibitor of phosphorylated ERK1/2, U0126 10 μM (A) or phosphorylated p38, SB202190 10 μM (B) . Representative Western blots from 3 independent experiments are shown and were used to assess the effect on CD248 expression. TGFβ-coupling to either ERK1/2 or to p38 is not involved in its suppressive effects on CD248.

    Article Snippet: The inhibitors SB431542 (for ALK5), SB202190 (for p38) and U0126 (for ERK1/2) were from Tocris Biosciences, Canada.

    Techniques: Incubation, Western Blot, Expressing

    Effects of p38 pathway in ouabain-induced effects in myogenic differentiation. Chick myogenic cells were grown for 24 h, treated with 10 μM ouabain and/or the p38 inhibitor SB202190 and fixed after 24 h. Cells were immunostained desmin (red) and DAPI (blue). Three independent experiments were performed and one representative image of each culture condition is shown. Scale bar = 50 μm.

    Journal: PLoS ONE

    Article Title: The Role of Na+/K+-ATPase during Chick Skeletal Myogenesis

    doi: 10.1371/journal.pone.0120940

    Figure Lengend Snippet: Effects of p38 pathway in ouabain-induced effects in myogenic differentiation. Chick myogenic cells were grown for 24 h, treated with 10 μM ouabain and/or the p38 inhibitor SB202190 and fixed after 24 h. Cells were immunostained desmin (red) and DAPI (blue). Three independent experiments were performed and one representative image of each culture condition is shown. Scale bar = 50 μm.

    Article Snippet: We also analyzed the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects by using the MEK-ERK inhibitor U0126 and the p38 inhibitor SB202190.

    Techniques:

    Phosphorylation of Sox9 and up-regulation of Sox9 protein by TGF-β1 is dependent on p38 activity. ( A , B ) ATDC5 cells were pretreated with different concentrations of SB203580 ( A ) and SB202190 ( B ). After 24 hours, cells were treated with vehicle control (−) or TGF-β1 (+) for 24 hours. Cell lysates were used in immunblots to measure p-MAPK-APK2 protein levels. N = 3. ( C ) Cells were pretreated with 10 μM SB203580 (p38i 1), 5 μM SB202190 (p38i 2), or DMSO control for 24 hours, and TGF-β1 (+) or vehicle control (−) was added to the cells, which were incubated for an additional 2 hours. Cell lysates were used in immunoblots to detect p-Sox9 protein, N = 4. ( D ) Cell lysates were also collected 6 hours after treatment with TGF-β1 (+) or vehicle control (−) and used to determine Sox9 protein levels, N = 6. ( E ) Sox9 mRNA levels were measured in cells that were pretreated with p38i 1, p38i 2, or DMSO and then treated with TGF-β1 or vehicle for 6 hours, N = 5. ( F ) ATDC5 cells were transfected with 30 pmol of p38α and p38β siRNA or N.S. siRNA. After 48 hours, cells were treated with TGF-β1 for 2 or 6 hours. Protein lysates were collected and p-Sox9 and Sox9 protein levels were determined by immunoblot, N = 3. ( G ) Papss2 mRNA levels were measured by QPCR after pretreatment with DMSO or p38i followed by treatment with TGF-β1 or vehicle. mRNA levels were determined relative to DMSO/vehicle treated samples. PPIA was used for gene expression normalization. All QPCR data was analyzed using REST software. *** p > 0.001, N = 4. Western blots were cropped for clarity. Examples of uncropped blots are found in Supplementary Figure S4 .

    Journal: Scientific Reports

    Article Title: TGF-β regulates phosphorylation and stabilization of Sox9 protein in chondrocytes through p38 and Smad dependent mechanisms

    doi: 10.1038/srep38616

    Figure Lengend Snippet: Phosphorylation of Sox9 and up-regulation of Sox9 protein by TGF-β1 is dependent on p38 activity. ( A , B ) ATDC5 cells were pretreated with different concentrations of SB203580 ( A ) and SB202190 ( B ). After 24 hours, cells were treated with vehicle control (−) or TGF-β1 (+) for 24 hours. Cell lysates were used in immunblots to measure p-MAPK-APK2 protein levels. N = 3. ( C ) Cells were pretreated with 10 μM SB203580 (p38i 1), 5 μM SB202190 (p38i 2), or DMSO control for 24 hours, and TGF-β1 (+) or vehicle control (−) was added to the cells, which were incubated for an additional 2 hours. Cell lysates were used in immunoblots to detect p-Sox9 protein, N = 4. ( D ) Cell lysates were also collected 6 hours after treatment with TGF-β1 (+) or vehicle control (−) and used to determine Sox9 protein levels, N = 6. ( E ) Sox9 mRNA levels were measured in cells that were pretreated with p38i 1, p38i 2, or DMSO and then treated with TGF-β1 or vehicle for 6 hours, N = 5. ( F ) ATDC5 cells were transfected with 30 pmol of p38α and p38β siRNA or N.S. siRNA. After 48 hours, cells were treated with TGF-β1 for 2 or 6 hours. Protein lysates were collected and p-Sox9 and Sox9 protein levels were determined by immunoblot, N = 3. ( G ) Papss2 mRNA levels were measured by QPCR after pretreatment with DMSO or p38i followed by treatment with TGF-β1 or vehicle. mRNA levels were determined relative to DMSO/vehicle treated samples. PPIA was used for gene expression normalization. All QPCR data was analyzed using REST software. *** p > 0.001, N = 4. Western blots were cropped for clarity. Examples of uncropped blots are found in Supplementary Figure S4 .

    Article Snippet: Cells were pretreated with p38 inhibitors SB203580 (Abcam), SB202190 (Abcam), or DMSO control for 24 hours before treatment with TGF-β1.

    Techniques: Activity Assay, Incubation, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, Software