Structured Review

Enzo Biochem sb 202190
Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or <t>SB-202190</t> ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P
Sb 202190, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sb 202190/product/Enzo Biochem
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
sb 202190 - by Bioz Stars, 2021-04
93/100 stars

Images

1) Product Images from "Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription"

Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

Journal: Scientific Reports

doi: 10.1038/s41598-020-68219-4

Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P
Figure Legend Snippet: Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P

Techniques Used: Activation Assay, Translocation Assay, Staining, Expressing, Western Blot, Transfection, Plasmid Preparation

SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. ( A , B ) SRC-3 is involved in NF-κB activation. ( A ) SRC-3 KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. ( B ) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. ( C , D ) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3 WT and SRC-3 KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 ( C ) and MMP9 ( D ) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3 WT cells. ( E , J ) MK2 and p38 MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 ( E ) or SB-202190 ( J ) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. ( F – H ) MK2 is involved in TNF-α induced TRAF1 ( F ), IL-8 ( G ) and ICAM1 ( H ) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. ( I ) p38 MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. ( K – M ) p38 MAPK is involved in TNF-α-induced IL-6 ( K ) and IL-8 ( L ) but not MMP9 ( M ) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; * P
Figure Legend Snippet: SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. ( A , B ) SRC-3 is involved in NF-κB activation. ( A ) SRC-3 KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. ( B ) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. ( C , D ) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3 WT and SRC-3 KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 ( C ) and MMP9 ( D ) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3 WT cells. ( E , J ) MK2 and p38 MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 ( E ) or SB-202190 ( J ) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. ( F – H ) MK2 is involved in TNF-α induced TRAF1 ( F ), IL-8 ( G ) and ICAM1 ( H ) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. ( I ) p38 MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. ( K – M ) p38 MAPK is involved in TNF-α-induced IL-6 ( K ) and IL-8 ( L ) but not MMP9 ( M ) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; * P

Techniques Used: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

Activation of p38 MAPK results in phosphorylation of SRC-3 at S857. ( A ) p38 MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38 MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. ( B – F ) p38 MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12 . H1299 ( B ), A549 ( C ), HEK 293 ( D ), HeLa ( E ) and MDA MB 231 ( F ) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38 MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The full-length blots are presented in supplementary figures S13 – S17 . ( G , H ) Inhibition of p38 MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin ( G ) or 500 μM sodium arsenite (SA) ( H ) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18 , S19 .
Figure Legend Snippet: Activation of p38 MAPK results in phosphorylation of SRC-3 at S857. ( A ) p38 MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38 MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. ( B – F ) p38 MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12 . H1299 ( B ), A549 ( C ), HEK 293 ( D ), HeLa ( E ) and MDA MB 231 ( F ) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38 MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The full-length blots are presented in supplementary figures S13 – S17 . ( G , H ) Inhibition of p38 MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin ( G ) or 500 μM sodium arsenite (SA) ( H ) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18 , S19 .

Techniques Used: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Inhibition

Related Articles

other:

Article Title: Nitric oxide induces cell death in canine cruciate ligament cells by activation of tyrosine kinase and reactive oxygen species
Article Snippet: Sodium nitroprusside (SNP), SB-202190, SN-50, and NS-398 were purchased from Enzo Life Sciences (Lausen, Switzerland).

Article Title: Human NDR Kinases Control G1/S Cell Cycle Transition by Directly Regulating p21 Stability ▿
Article Snippet: Okadaic acid (OA), SB203580, and SB202190 were from Alexis (Enzo Life Sciences).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Enzo Biochem p38 map kinase inhibitor sb202190
    Effects of SOCS1 on <t>MAP</t> kinase signaling. SW1353 cells were transfected with SOCS1 or shSOCS1 vectors to overexpress or inhibit SOCS1, as described in Methods. A representative immunoblot image showed that SOCS1 overexpression decreased phosphorylation of <t>p38</t> and JNK, whereas SOCS1 knockdown increased their phosphorylation in the presence of IL-1β (10 ng/ml, A) . The relative proportions of phosphorylated to total protein were determined with densitometry by using Image J software (version 1.48c, [ 22 ]; B) . Data were expressed as the means ± SEM ( n = 3). OE, overexpression; KD, knockdown.
    P38 Map Kinase Inhibitor Sb202190, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p38 map kinase inhibitor sb202190/product/Enzo Biochem
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p38 map kinase inhibitor sb202190 - by Bioz Stars, 2021-04
    86/100 stars
      Buy from Supplier

    93
    Enzo Biochem sb202190
    Luteolin blocked P38 phosphorylation, and the P38 inhibitor <t>SB202190</t> (SB) decreased THP-1 cell adherence to IL-1 β -stimulated ARPE-19 cells. (a) Western blots show levels of phosphorylated P38 protein expression. (b) The fold-change in pP38 protein expression was measured relative to P38 expression. (c) ARPE-19 cells were pretreated with 10 μ M luteolin or P38 inhibitor (SB203580) for 1 h and then cocultured with labeled THP-1 cells. (d) The fluorescence intensity was used to quantify calcein-AM fluorescence. Data represent the mean ± SD. ∗ p
    Sb202190, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb202190/product/Enzo Biochem
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sb202190 - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

    93
    Enzo Biochem p38 mapk inhibitor sb202190
    Proposed mechanisms of PGE 2 production induced by <t>TLR2/p38</t> <t>MAPK</t> and EP4 stimulation, leading to optimal PGE 2 production and protection against necrosis in Mtb -infected Mφs. PGE 2 generated by TLR2 stimulation/p38 MAPK phosphorylation following
    P38 Mapk Inhibitor Sb202190, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p38 mapk inhibitor sb202190/product/Enzo Biochem
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p38 mapk inhibitor sb202190 - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

    93
    Enzo Biochem sb 202190
    Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or <t>SB-202190</t> ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P
    Sb 202190, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sb 202190/product/Enzo Biochem
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sb 202190 - by Bioz Stars, 2021-04
    93/100 stars
      Buy from Supplier

    Image Search Results


    Effects of SOCS1 on MAP kinase signaling. SW1353 cells were transfected with SOCS1 or shSOCS1 vectors to overexpress or inhibit SOCS1, as described in Methods. A representative immunoblot image showed that SOCS1 overexpression decreased phosphorylation of p38 and JNK, whereas SOCS1 knockdown increased their phosphorylation in the presence of IL-1β (10 ng/ml, A) . The relative proportions of phosphorylated to total protein were determined with densitometry by using Image J software (version 1.48c, [ 22 ]; B) . Data were expressed as the means ± SEM ( n = 3). OE, overexpression; KD, knockdown.

    Journal: Arthritis Research & Therapy

    Article Title: Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

    doi: 10.1186/ar4381

    Figure Lengend Snippet: Effects of SOCS1 on MAP kinase signaling. SW1353 cells were transfected with SOCS1 or shSOCS1 vectors to overexpress or inhibit SOCS1, as described in Methods. A representative immunoblot image showed that SOCS1 overexpression decreased phosphorylation of p38 and JNK, whereas SOCS1 knockdown increased their phosphorylation in the presence of IL-1β (10 ng/ml, A) . The relative proportions of phosphorylated to total protein were determined with densitometry by using Image J software (version 1.48c, [ 22 ]; B) . Data were expressed as the means ± SEM ( n = 3). OE, overexpression; KD, knockdown.

    Article Snippet: A p38 MAP kinase inhibitor SB202190 and NF-κB inhibitor SN50 were purchased from Alexis Biochemicals (Farmingdale, MI, USA).

    Techniques: Transfection, Over Expression, Software

    Effect of MAP kinase and NF-κB inhibitors on MMPs secretion from SOCS1-knockdown SW1353 cells. SOCS1-knockdown SW1353 cells were pretreated with inhibitors for 1 hour before stimulation with 10 ng/ml of IL-1β. After 24 hours, the levels of MMP-1, -3, and -13 were dramatically decreased by SB202190, a p38 MAP kinase inhibitor (A) . Blockade of C-JNK (B) and ERK (C) dose-dependently suppressed the secretion of MMPs from shSOCS1-transfected SW1353 cells. The production of MMP-1 and MMP-13 was partially inhibited with the SN50, a specific NF-κB inhibitory peptide (D) . Data were expressed as the mean ± SEM of relative MMPs levels, as compared with the control without inhibitors ( n = 3). *P

    Journal: Arthritis Research & Therapy

    Article Title: Cytokine signaling-1 suppressor is inducible by IL-1beta and inhibits the catabolic effects of IL-1beta in chondrocytes: its implication in the paradoxical joint-protective role of IL-1beta

    doi: 10.1186/ar4381

    Figure Lengend Snippet: Effect of MAP kinase and NF-κB inhibitors on MMPs secretion from SOCS1-knockdown SW1353 cells. SOCS1-knockdown SW1353 cells were pretreated with inhibitors for 1 hour before stimulation with 10 ng/ml of IL-1β. After 24 hours, the levels of MMP-1, -3, and -13 were dramatically decreased by SB202190, a p38 MAP kinase inhibitor (A) . Blockade of C-JNK (B) and ERK (C) dose-dependently suppressed the secretion of MMPs from shSOCS1-transfected SW1353 cells. The production of MMP-1 and MMP-13 was partially inhibited with the SN50, a specific NF-κB inhibitory peptide (D) . Data were expressed as the mean ± SEM of relative MMPs levels, as compared with the control without inhibitors ( n = 3). *P

    Article Snippet: A p38 MAP kinase inhibitor SB202190 and NF-κB inhibitor SN50 were purchased from Alexis Biochemicals (Farmingdale, MI, USA).

    Techniques: Transfection

    Luteolin blocked P38 phosphorylation, and the P38 inhibitor SB202190 (SB) decreased THP-1 cell adherence to IL-1 β -stimulated ARPE-19 cells. (a) Western blots show levels of phosphorylated P38 protein expression. (b) The fold-change in pP38 protein expression was measured relative to P38 expression. (c) ARPE-19 cells were pretreated with 10 μ M luteolin or P38 inhibitor (SB203580) for 1 h and then cocultured with labeled THP-1 cells. (d) The fluorescence intensity was used to quantify calcein-AM fluorescence. Data represent the mean ± SD. ∗ p

    Journal: Mediators of Inflammation

    Article Title: Luteolin Attenuates IL-1β-Induced THP-1 Adhesion to ARPE-19 Cells via Suppression of NF-κB and MAPK Pathways

    doi: 10.1155/2020/9421340

    Figure Lengend Snippet: Luteolin blocked P38 phosphorylation, and the P38 inhibitor SB202190 (SB) decreased THP-1 cell adherence to IL-1 β -stimulated ARPE-19 cells. (a) Western blots show levels of phosphorylated P38 protein expression. (b) The fold-change in pP38 protein expression was measured relative to P38 expression. (c) ARPE-19 cells were pretreated with 10 μ M luteolin or P38 inhibitor (SB203580) for 1 h and then cocultured with labeled THP-1 cells. (d) The fluorescence intensity was used to quantify calcein-AM fluorescence. Data represent the mean ± SD. ∗ p

    Article Snippet: The inhibitors PD98059, SP600125, SB202190, and Bay 117082 were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Western Blot, Expressing, Labeling, Fluorescence

    Proposed mechanisms of PGE 2 production induced by TLR2/p38 MAPK and EP4 stimulation, leading to optimal PGE 2 production and protection against necrosis in Mtb -infected Mφs. PGE 2 generated by TLR2 stimulation/p38 MAPK phosphorylation following

    Journal: The FASEB Journal

    Article Title: The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis

    doi: 10.1096/fj.13-228858

    Figure Lengend Snippet: Proposed mechanisms of PGE 2 production induced by TLR2/p38 MAPK and EP4 stimulation, leading to optimal PGE 2 production and protection against necrosis in Mtb -infected Mφs. PGE 2 generated by TLR2 stimulation/p38 MAPK phosphorylation following

    Article Snippet: Phospho-p38 mitogen-activated protein kinase (MAPK; Thr180 /Tyr182 ) antibody, p38 MAPK antibody, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 14C10) rabbit mAb, and COX1 (D2G6) rabbit mAb were from Cell Signaling Technology (Beverly, MA, USA); p38 MAPK inhibitor SB202190 was from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Infection, Generated

    Regulation of COX2 and mPGES-1 expression and PGE 2 production by p38 MAPK in H37Ra-infected human Mφs. A ) Western blot of time-dependent p38 MAPK phosphorylation in H37Ra infection (MOI 10:1). Ratio of P-p38 MAPK to p38 MAPK is indicated for every

    Journal: The FASEB Journal

    Article Title: The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis

    doi: 10.1096/fj.13-228858

    Figure Lengend Snippet: Regulation of COX2 and mPGES-1 expression and PGE 2 production by p38 MAPK in H37Ra-infected human Mφs. A ) Western blot of time-dependent p38 MAPK phosphorylation in H37Ra infection (MOI 10:1). Ratio of P-p38 MAPK to p38 MAPK is indicated for every

    Article Snippet: Phospho-p38 mitogen-activated protein kinase (MAPK; Thr180 /Tyr182 ) antibody, p38 MAPK antibody, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 14C10) rabbit mAb, and COX1 (D2G6) rabbit mAb were from Cell Signaling Technology (Beverly, MA, USA); p38 MAPK inhibitor SB202190 was from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Expressing, Infection, Western Blot

    TLR2 stimulation of human Mφs induces COX2 and mPGES-1 expression through the p38 MAPK and EP4 signaling pathways. A ) Western blot of p38 MAPK phosphorylation at different time points in Mφs treated with TLR1/2 agonist Pam3CSK4 (10 μg/ml)

    Journal: The FASEB Journal

    Article Title: The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis

    doi: 10.1096/fj.13-228858

    Figure Lengend Snippet: TLR2 stimulation of human Mφs induces COX2 and mPGES-1 expression through the p38 MAPK and EP4 signaling pathways. A ) Western blot of p38 MAPK phosphorylation at different time points in Mφs treated with TLR1/2 agonist Pam3CSK4 (10 μg/ml)

    Article Snippet: Phospho-p38 mitogen-activated protein kinase (MAPK; Thr180 /Tyr182 ) antibody, p38 MAPK antibody, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 14C10) rabbit mAb, and COX1 (D2G6) rabbit mAb were from Cell Signaling Technology (Beverly, MA, USA); p38 MAPK inhibitor SB202190 was from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Expressing, Western Blot

    Role of EP4 in the activation of the p38 MAPK signaling in H37Ra-infected human Mφs. A ) Western blot of p38 MAPK phosphorylation in Mφs stimulated with the EP4 agonist ONO-AE1-329 (10 μM). Mφ lysates treated with DMSO (final

    Journal: The FASEB Journal

    Article Title: The prostaglandin E2 receptor EP4 is integral to a positive feedback loop for prostaglandin E2 production in human macrophages infected with Mycobacterium tuberculosis

    doi: 10.1096/fj.13-228858

    Figure Lengend Snippet: Role of EP4 in the activation of the p38 MAPK signaling in H37Ra-infected human Mφs. A ) Western blot of p38 MAPK phosphorylation in Mφs stimulated with the EP4 agonist ONO-AE1-329 (10 μM). Mφ lysates treated with DMSO (final

    Article Snippet: Phospho-p38 mitogen-activated protein kinase (MAPK; Thr180 /Tyr182 ) antibody, p38 MAPK antibody, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 14C10) rabbit mAb, and COX1 (D2G6) rabbit mAb were from Cell Signaling Technology (Beverly, MA, USA); p38 MAPK inhibitor SB202190 was from Enzo Life Sciences (Farmingdale, NY, USA).

    Techniques: Activation Assay, Infection, Western Blot

    Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P

    Journal: Scientific Reports

    Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

    doi: 10.1038/s41598-020-68219-4

    Figure Lengend Snippet: Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or SB-202190 ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P

    Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

    Techniques: Activation Assay, Translocation Assay, Staining, Expressing, Western Blot, Transfection, Plasmid Preparation

    SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. ( A , B ) SRC-3 is involved in NF-κB activation. ( A ) SRC-3 KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. ( B ) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. ( C , D ) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3 WT and SRC-3 KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 ( C ) and MMP9 ( D ) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3 WT cells. ( E , J ) MK2 and p38 MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 ( E ) or SB-202190 ( J ) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. ( F – H ) MK2 is involved in TNF-α induced TRAF1 ( F ), IL-8 ( G ) and ICAM1 ( H ) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. ( I ) p38 MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. ( K – M ) p38 MAPK is involved in TNF-α-induced IL-6 ( K ) and IL-8 ( L ) but not MMP9 ( M ) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; * P

    Journal: Scientific Reports

    Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

    doi: 10.1038/s41598-020-68219-4

    Figure Lengend Snippet: SRC-3 is required for MK2-mediated induction of IL-6 expression in response to TNF-α. ( A , B ) SRC-3 is involved in NF-κB activation. ( A ) SRC-3 KO A549 cells were co-transfected with 120 ng κB-ConA-luc vector and 50 ng of either pSG5 empty vector, SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG vector. After 48 h TNF-α was added (if not other indicated 10 ng/ml for 5 h) before determination of luciferase activity relative to pSG5. ( B ) A549-NF-κB-Luc cells were transfected with scrambled siRNA or SRC-3 siRNA and 48 h later stimulated with TNF-α or left unstimulated. Nontransfected cells were pretreated with PF-3644022 for 30 min before TNF-α treatment. Luciferase activities are shown relative to unstimulated scrambled siRNA. ( C , D ) SRC-3 is involved in TNF-α-induced IL-6 expression. SRC-3 WT and SRC-3 KO A549 cells were stimulated with TNF-α for 2 h or left unstimulated. mRNA expression of IL-6 ( C ) and MMP9 ( D ) were determined relative to GAPDH and TFRC. Fold changes are presented relative to unstimulated SRC-3 WT cells. ( E , J ) MK2 and p38 MAPK activity are required for transcription of IL-6. A549 cells were transfected with 120 ng pGL3-IL-6-promoter vector and after 48 h treated for 30 min with 0.2 μl DMSO, 10 μM PF-3644022 ( E ) or SB-202190 ( J ) followed by stimulation with TNF-α. Luciferase activities are shown relative to DMSO. ( F – H ) MK2 is involved in TNF-α induced TRAF1 ( F ), IL-8 ( G ) and ICAM1 ( H ) mRNA expression. A549 cells pretreated with DMSO or 10 μM PF-3644022 for 30 min were stimulated with TNF-α for 2 h or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. ( I ) p38 MAPK is involved in NF-κB-dependent luciferase activity. A549-NF-κB-Luc cells were pretreated with DMSO or SB-202190 and then stimulated with TNF-α or left unstimulated. Luciferase activities are shown relative to DMSO. ( K – M ) p38 MAPK is involved in TNF-α-induced IL-6 ( K ) and IL-8 ( L ) but not MMP9 ( M ) mRNA expression. A549 cells pretreated with DMSO or 10 μM SB-202190 for 30 min were stimulated with TNF-α or left unstimulated. MRNA expression were determined relative to GAPDH and TFRC. Fold changes are presented relative to DMSO. Data are presented as mean ± SD (n = 3). Unpaired t-test; * P

    Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

    Techniques: Expressing, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    Activation of p38 MAPK results in phosphorylation of SRC-3 at S857. ( A ) p38 MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38 MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. ( B – F ) p38 MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12 . H1299 ( B ), A549 ( C ), HEK 293 ( D ), HeLa ( E ) and MDA MB 231 ( F ) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38 MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The full-length blots are presented in supplementary figures S13 – S17 . ( G , H ) Inhibition of p38 MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin ( G ) or 500 μM sodium arsenite (SA) ( H ) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18 , S19 .

    Journal: Scientific Reports

    Article Title: Phosphorylation of steroid receptor coactivator-3 (SRC-3) at serine 857 is regulated by the p38MAPK-MK2 axis and affects NF-κB-mediated transcription

    doi: 10.1038/s41598-020-68219-4

    Figure Lengend Snippet: Activation of p38 MAPK results in phosphorylation of SRC-3 at S857. ( A ) p38 MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38 MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. ( B – F ) p38 MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12 . H1299 ( B ), A549 ( C ), HEK 293 ( D ), HeLa ( E ) and MDA MB 231 ( F ) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38 MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38 MAPK and anti-p38 MAPK antibodies. The full-length blots are presented in supplementary figures S13 – S17 . ( G , H ) Inhibition of p38 MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin ( G ) or 500 μM sodium arsenite (SA) ( H ) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18 , S19 .

    Article Snippet: SB-202190 (#BML-EI294-001), PD-184352 (#ALX-270-471) were purchased from Alexis Biochemicals, CA, USA.

    Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Multiple Displacement Amplification, Inhibition