anti-jnk Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc anti jnk
    Analysis of pathways involved in mig-2-mediated cisplatin-induced apoptosis in H4 cells. Ectopic expression or knock-down of mig-2 in H4 cells treated with 40 μmol/L of cisplatin for 24 h. (A) Western blotting analysis of the total expression and activation of ERK1/2, <t>JNK,</t> p38, <t>ERK5</t> and AKT. (B) Analysis of mig-2-mediated cisplatin-induced apoptosis when cells were pretreated with JNK inhibitor (SP600125, SP), p38 inhibitor (SB203580, SB) or AKT inhibitor (LY294002, LY) for 1 h. The data are presented as the mean±SD from three independent experiments. b P
    Anti Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 5368 article reviews
    Price from $9.99 to $1999.99
    anti jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti phospho jnk
    Analysis of pathways involved in mig-2-mediated cisplatin-induced apoptosis in H4 cells. Ectopic expression or knock-down of mig-2 in H4 cells treated with 40 μmol/L of cisplatin for 24 h. (A) Western blotting analysis of the total expression and activation of ERK1/2, <t>JNK,</t> p38, <t>ERK5</t> and AKT. (B) Analysis of mig-2-mediated cisplatin-induced apoptosis when cells were pretreated with JNK inhibitor (SP600125, SP), p38 inhibitor (SB203580, SB) or AKT inhibitor (LY294002, LY) for 1 h. The data are presented as the mean±SD from three independent experiments. b P
    Anti Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 2709 article reviews
    Price from $9.99 to $1999.99
    anti phospho jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc phospho jnk
    Analysis of pathways involved in mig-2-mediated cisplatin-induced apoptosis in H4 cells. Ectopic expression or knock-down of mig-2 in H4 cells treated with 40 μmol/L of cisplatin for 24 h. (A) Western blotting analysis of the total expression and activation of ERK1/2, <t>JNK,</t> p38, <t>ERK5</t> and AKT. (B) Analysis of mig-2-mediated cisplatin-induced apoptosis when cells were pretreated with JNK inhibitor (SP600125, SP), p38 inhibitor (SB203580, SB) or AKT inhibitor (LY294002, LY) for 1 h. The data are presented as the mean±SD from three independent experiments. b P
    Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phospho jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 6198 article reviews
    Price from $9.99 to $1999.99
    phospho jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc anti p jnk
    The inhibition of <t>JNK</t> and Nod1 reduced the expression of <t>TNFα</t> and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p
    Anti P Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1497 article reviews
    Price from $9.99 to $1999.99
    anti p jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    93
    Santa Cruz Biotechnology anti jnk
    PR55γ Is a Regulator of <t>JNK</t> following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. <t>GFP</t> expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002
    Anti Jnk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti jnk/product/Santa Cruz Biotechnology
    Average 93 stars, based on 980 article reviews
    Price from $9.99 to $1999.99
    anti jnk - by Bioz Stars, 2020-10
    93/100 stars
      Buy from Supplier

    89
    Santa Cruz Biotechnology anti phosphorylated jnk
    Up-regulated HRAS increased phosphorylated ERK1/2, <t>JNK,</t> and HSP27 and promoted cell motility and invasiveness. A , scrambled control siRNA oligonucleotides ( Scr )-transfected S2-013 cells and siRNA oligonucleotides targeting HRAS ( siHRAS )-transfected S2-013 cells were incubated on fibronectin. Western blotting ( IB ) was performed using the indicated antibodies. B , a <t>Myc-tagged</t> HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with scrambled control siRNA or HOXB7 siRNA with or without HRAS siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies. C , transwell motility and invasion assays. Oligonucleotides (siRNAs targeting HRAS or scrambled siRNAs as the negative control) were transiently transfected into S2-013 cells; 48 h later, transwell motility and Matrigel invasion assays were performed. Migrating cells in four visual fields per group were scored. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p
    Anti Phosphorylated Jnk, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 89/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phosphorylated jnk/product/Santa Cruz Biotechnology
    Average 89 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    anti phosphorylated jnk - by Bioz Stars, 2020-10
    89/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc rabbit anti phospho jnk
    Up-regulated HRAS increased phosphorylated ERK1/2, <t>JNK,</t> and HSP27 and promoted cell motility and invasiveness. A , scrambled control siRNA oligonucleotides ( Scr )-transfected S2-013 cells and siRNA oligonucleotides targeting HRAS ( siHRAS )-transfected S2-013 cells were incubated on fibronectin. Western blotting ( IB ) was performed using the indicated antibodies. B , a <t>Myc-tagged</t> HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with scrambled control siRNA or HOXB7 siRNA with or without HRAS siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies. C , transwell motility and invasion assays. Oligonucleotides (siRNAs targeting HRAS or scrambled siRNAs as the negative control) were transiently transfected into S2-013 cells; 48 h later, transwell motility and Matrigel invasion assays were performed. Migrating cells in four visual fields per group were scored. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p
    Rabbit Anti Phospho Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 373 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti phospho jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 373 article reviews
    Price from $9.99 to $1999.99
    rabbit anti phospho jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    92
    Santa Cruz Biotechnology anti jnk1
    Structure of <t>JNK1</t> in complex with MKP7-CD. ( a ) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. ( b ) Structure of MKP7-CD with its active site highlight in cyan. The 2 F o − F c omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b . ( c ) Structure of VHR with its active site highlighted in marine blue. ( d ) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). ( e ) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). Blue dashed lines represent polar interactions. ( f ) The 2 F o − F c omit map (contoured at 1.5σ) clearly shows electron density for the 285 FNFL 288 segment of MKP7-CD.
    Anti Jnk1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti jnk1/product/Santa Cruz Biotechnology
    Average 92 stars, based on 436 article reviews
    Price from $9.99 to $1999.99
    anti jnk1 - by Bioz Stars, 2020-10
    92/100 stars
      Buy from Supplier

    90
    Cell Signaling Technology Inc anti phospho jnk1 2
    Resveratrol inhibits S. aureus -induced VCAM-1 expression and inflammatory signaling pathways. ( A ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. ( B ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 24 h. The THP-1 cells adherence was measured. ( C – E ) Cells were pretreated without or with resveratrol for 24 h, and then incubated with S. aureus for the indicated times. The expression of phospho-c-Src, phospho-PDGFR, phospho-p38 MAPK, <t>phospho-JNK1/2,</t> phospho-c-Jun, and phospho-ATF2 were determined by Western blot. ( F ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h. ATF2 and c-Jun binding activities were analyzed by a ChIP assay. n = 3–4, # p
    Anti Phospho Jnk1 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti phospho jnk1 2/product/Cell Signaling Technology Inc
    Average 90 stars, based on 226 article reviews
    Price from $9.99 to $1999.99
    anti phospho jnk1 2 - by Bioz Stars, 2020-10
    90/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc p jnk
    IL-1β induces linker phosphorylation of <t>Smad3</t> in HSCs. (A) HSCs-P1 were treated with R-III (0.3 μM) for 20 h and cell lysates were analyzed by western blotting. The Western blots are representative of three independent experiments from separate cell preparations. α-tubulin was used as a loading control. (B) HSCs-P1 were treated with R-III in the presence of IL-1RA (1 μg/ml) and analyzed by western blotting. (C, D) HSCs-P1 were treated with R-III in the presence of the inhibitor for TAK1 ((5Z)-7-Oxozeaenol, 0.25 μM), p38 (SB203580, 5 μM) and <t>JNK</t> (SP600125, 10 μM) and analyzed by western blotting (C) and real-time PCR (D). P -value, paired t-test (n = 3) (compared to untreated HSCs-P1), *P
    P Jnk, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 7240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p jnk/product/Cell Signaling Technology Inc
    Average 99 stars, based on 7240 article reviews
    Price from $9.99 to $1999.99
    p jnk - by Bioz Stars, 2020-10
    99/100 stars
      Buy from Supplier

    Image Search Results


    Analysis of pathways involved in mig-2-mediated cisplatin-induced apoptosis in H4 cells. Ectopic expression or knock-down of mig-2 in H4 cells treated with 40 μmol/L of cisplatin for 24 h. (A) Western blotting analysis of the total expression and activation of ERK1/2, JNK, p38, ERK5 and AKT. (B) Analysis of mig-2-mediated cisplatin-induced apoptosis when cells were pretreated with JNK inhibitor (SP600125, SP), p38 inhibitor (SB203580, SB) or AKT inhibitor (LY294002, LY) for 1 h. The data are presented as the mean±SD from three independent experiments. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways

    doi: 10.1038/aps.2014.60

    Figure Lengend Snippet: Analysis of pathways involved in mig-2-mediated cisplatin-induced apoptosis in H4 cells. Ectopic expression or knock-down of mig-2 in H4 cells treated with 40 μmol/L of cisplatin for 24 h. (A) Western blotting analysis of the total expression and activation of ERK1/2, JNK, p38, ERK5 and AKT. (B) Analysis of mig-2-mediated cisplatin-induced apoptosis when cells were pretreated with JNK inhibitor (SP600125, SP), p38 inhibitor (SB203580, SB) or AKT inhibitor (LY294002, LY) for 1 h. The data are presented as the mean±SD from three independent experiments. b P

    Article Snippet: Antibodies and special reagents Cleaved caspase-3 antibody, cleaved caspase-8 antibody, cleaved caspase-9 antibody, cleaved PARP antibody, Bid antibody, Bax antibody, Bcl-2 antibody, cytochrome c antibody, AKT antibody, p-Akt (Ser473) antibody, ERK5 antibody, p-ERK5 (Thr218/Tyr220) antibody, ERK1/2 antibody, p-ERK1/2 (Thr202/Tyr204) antibody, JNK antibody, p-JNK antibody (Thr183/Tyr185), p38 antibody, p-p38 (Thr180/Tyr182) antibody, GFP antibody and β-actin antibody were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Expressing, Western Blot, Activation Assay

    F3 subdomain of mig-2 is necessary and sufficient for mig-2 antagonizing cisplatin-induced apoptosis. (A) The ectopic expression of mutant and wild-type mig-2 in the glioma cells line H4 was confirmed by Western blotting using an anti-GFP antibody (arrows). (B) Analysis of cisplatin-induced apoptosis in mutants or wild-type mig-2 cells using Annexin V/PI staining. The bar chart shows the percentage of apoptotic cells (early apoptotic+late apoptotic). (C) Western blotting analysis of cleaved-PARP, cleaved-caspase-8, cleaved-caspase-9, cleaved-caspase-3, activation of Bid, Bax, Bcl-2, and cytochrome c . The total expression and activation of JNK, p38, and AKT after cells were treated or not treated with cisplatin for 24 h in mutant or wild-type mig-2 cells. The data are presented as the mean±SD from three independent experiments. b P

    Journal: Acta Pharmacologica Sinica

    Article Title: Mig-2 attenuates cisplatin-induced apoptosis of human glioma cells in vitro through AKT/JNK and AKT/p38 signaling pathways

    doi: 10.1038/aps.2014.60

    Figure Lengend Snippet: F3 subdomain of mig-2 is necessary and sufficient for mig-2 antagonizing cisplatin-induced apoptosis. (A) The ectopic expression of mutant and wild-type mig-2 in the glioma cells line H4 was confirmed by Western blotting using an anti-GFP antibody (arrows). (B) Analysis of cisplatin-induced apoptosis in mutants or wild-type mig-2 cells using Annexin V/PI staining. The bar chart shows the percentage of apoptotic cells (early apoptotic+late apoptotic). (C) Western blotting analysis of cleaved-PARP, cleaved-caspase-8, cleaved-caspase-9, cleaved-caspase-3, activation of Bid, Bax, Bcl-2, and cytochrome c . The total expression and activation of JNK, p38, and AKT after cells were treated or not treated with cisplatin for 24 h in mutant or wild-type mig-2 cells. The data are presented as the mean±SD from three independent experiments. b P

    Article Snippet: Antibodies and special reagents Cleaved caspase-3 antibody, cleaved caspase-8 antibody, cleaved caspase-9 antibody, cleaved PARP antibody, Bid antibody, Bax antibody, Bcl-2 antibody, cytochrome c antibody, AKT antibody, p-Akt (Ser473) antibody, ERK5 antibody, p-ERK5 (Thr218/Tyr220) antibody, ERK1/2 antibody, p-ERK1/2 (Thr202/Tyr204) antibody, JNK antibody, p-JNK antibody (Thr183/Tyr185), p38 antibody, p-p38 (Thr180/Tyr182) antibody, GFP antibody and β-actin antibody were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

    Techniques: Expressing, Mutagenesis, Western Blot, Staining, Activation Assay

    Activation of ERK and p38 MAPK but not JNK signaling pathways by AGE-BSA . Representative western blots of ERK, phosphorylated ERK, p38 MAPK, phosphorylated p38 MAPK, JNK, and phosphorylated JNK in cells treated with AGE-BSA for 2 (A) and 6 hours (B). Phosphorylation of ERK and p38 MAPK was activated dose-dependently but no such effect was seen for JNK.

    Journal: BMC Cell Biology

    Article Title: AGE-BSA down-regulates endothelial connexin43 gap junctions

    doi: 10.1186/1471-2121-12-19

    Figure Lengend Snippet: Activation of ERK and p38 MAPK but not JNK signaling pathways by AGE-BSA . Representative western blots of ERK, phosphorylated ERK, p38 MAPK, phosphorylated p38 MAPK, JNK, and phosphorylated JNK in cells treated with AGE-BSA for 2 (A) and 6 hours (B). Phosphorylation of ERK and p38 MAPK was activated dose-dependently but no such effect was seen for JNK.

    Article Snippet: The membrane was incubated with the monoclonal anti-Cx43 antibody (1:1000; Chemicon), anti-Cx43 antibody (1:1000; BD Biosciences), anti-β actin antibody (1:2000; Chemicon), anti-ERK antibody (1:1000; Cell signaling), anti-p38 MAPK antibody (1:1000; Biosource), anti-JNK antibody (1:2000; Cell signaling), anti-phosphorylated ERK antibody (1:1000; Cell signaling), anti-phosphorylated p38 MAPK antibody (1:1000; Biosource), and anti-phosphorylated JNK antibody (1:1000; Cell signaling) at room temperature for 1 hour.

    Techniques: Activation Assay, Western Blot

    Role of the JNK1/2 MAPK pathway in LPS potentiation of TGFβ1-induced PDGF-B mRNA expression in MMNK-1 cells

    Journal: Journal of Gastroenterology and Hepatology

    Article Title: Lipopolysaccharide enhances transforming growth factor ?1-induced PDGF-B expression in bile duct epithelial cells

    doi: 10.1111/j.1440-1746.2011.06941.x

    Figure Lengend Snippet: Role of the JNK1/2 MAPK pathway in LPS potentiation of TGFβ1-induced PDGF-B mRNA expression in MMNK-1 cells

    Article Snippet: The membranes were blocked for 1 hr at room temperature in 3% BSA in TBST and incubated overnight at 4°C in either anti-IκBα (1:1000) antibody, anti-GAPDH (1:10000) antibody, anti-phospho-JNK1/2 (1:1000) antibody, or anti-JNK1/2 (1:2000) antibody (Cell Signaling Technology, Inc., Danvers, MA) in 1% BSA in TBST.

    Techniques: Expressing

    The inhibition of JNK and Nod1 reduced the expression of TNFα and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p

    Journal: Biochemical and biophysical research communications

    Article Title: TNFα and IL-1β are mediated by both TLR4 and Nod1 pathways in the cultured HAPI cells stimulated by LPS

    doi: 10.1016/j.bbrc.2012.03.068

    Figure Lengend Snippet: The inhibition of JNK and Nod1 reduced the expression of TNFα and IL-1β in HAPI cells treated with LPS. HAPI cells were pretreated with SP600125 (SP, JNK inhibitor) for 1 h, then treated with LPS. SP600125 treatment reduced the expression of TNFα (A), and IL-1β (B), ** p

    Article Snippet: The membrane was then blocked and then incubated with primary antibodies overnight at 4°C, including rabbit anti-pNF-κB (1:2000, Cell Signaling, Danvers, MA), mouse anti-NF-κB(1:2000, Millipore, Billerica, MA), mouse anti-pJNK (1:2000, Cell Signaling), rabbit anti-JNK (1:2000, Cell Signaling), rabbit anti-TNFα (1:1000, Millipore, Billerica, MA), rabbit anti-IL-1β (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-TLR-4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), and mouse anti-β-actin (1:10000, Santa Cruz Biotechnology).

    Techniques: Inhibition, Expressing

    PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Is a Regulator of JNK following UV Irradiation (A) U2-OS cells expressing pcDNA-HA-PR55γ were cotransfected with the pooled knockdown (PR55γ KD ) vectors as indicated (A–E) or a control vector. GFP expression serves as a measure of transfection consistency. (B) U2-OS cells were cotransfected with PR55γ KD vectors #1 or #2, pcDNA-PR55γ serves as a positive control. pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. mRNA levels relative to the control are shown as evaluated by quantitative real-time PCR. (C) U2-OS cells were cotransfected with PR55γ KD vectors as indicated (#1 or #2) or control vector. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells expressing pcDNA-HA-PR55γ or pcDNA-HA-PR55γ (Δ) were cotransfected with PR55γ KD vector #2. Protein samples were analyzed by immunoblotting with antibodies targeting HA. (E) U2-OS cells expressing pcDNA-HA-PR55γ, pcDNA-HA- PR55γ(Δ), or a control vector were cotransfected with PR55γ KD vectors #1 or #2. A pSuper vector targeting a mouse PP2A subunit PR59 served as an shRNA control. Selected cells were exposed to UV irradiation (100 J/m 2 ) and incubated for a further 60 min. Protein samples were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), total JNK (α-JNK), or haemoglutinin (α-HA, reprobe). (F) U2-OS cells expressing PR55γ KD2 vector or a control vector exposed to TNF-α, EGF, NaCl, or insulin for 5 min and incubated for a further 30–60 minutes. pJNK relative to total JNK levels are shown. doi:10.1371/journal.pgen.0030218.g002

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Irradiation, Expressing, Plasmid Preparation, Transfection, Positive Control, shRNA, Real-time Polymerase Chain Reaction, Incubation

    Model for the Regulation of c-SRC-Induced JNK Activation following UV Signaling by PP2A Complexes doi:10.1371/journal.pgen.0030218.g009

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: Model for the Regulation of c-SRC-Induced JNK Activation following UV Signaling by PP2A Complexes doi:10.1371/journal.pgen.0030218.g009

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Activation Assay

    Inhibition of JNK Activity by PR55γ Is Dependent upon Ser12 of c-SRC (A) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12A . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK), JNK1 and JNK2 (α-JNK), or FLAG (α-FLAG). (B) GFP-PR55γ vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12D . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), FLAG (α-FLAG), or GFP (α-GFP). (C) U2-OS cells coexpressing PR55γ shRNAs and FLAG-SRC or FLAG- SRC S12A as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. (D) U2-OS cells expressing PR55γ and FLAG-SRC or FLAG- SRC S12D as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. doi:10.1371/journal.pgen.0030218.g008

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: Inhibition of JNK Activity by PR55γ Is Dependent upon Ser12 of c-SRC (A) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12A . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK), JNK1 and JNK2 (α-JNK), or FLAG (α-FLAG). (B) GFP-PR55γ vector or control vector were cotransfected as indicated in the presence of FLAG-SRC or FLAG- SRC S12D . Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), FLAG (α-FLAG), or GFP (α-GFP). (C) U2-OS cells coexpressing PR55γ shRNAs and FLAG-SRC or FLAG- SRC S12A as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. (D) U2-OS cells expressing PR55γ and FLAG-SRC or FLAG- SRC S12D as indicated. Selected cells were exposed to UV irradiation (50 J/m 2 ) and treated 18 h later with fluorescent dye measuring mitochondria membrane potential (DiOC6[ 3 ]). Figure represents three independent experiments. doi:10.1371/journal.pgen.0030218.g008

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Inhibition, Activity Assay, Plasmid Preparation, Irradiation, Expressing

    PR55γ Regulates JNK Upstream of MKK4 and at the Level or Upstream of c-SRC (A) pJNK and pMKK4 in relation to the total level of unphosphorylated protein in UV irradiated U2-OS cells followed over time (0–60 min) in the presence or absence of PR55γ KD2 vector. (B) U2-OS cells expressing PR55γ KD2 or a control vector were treated with UV and incubated for 60 min. Whole cell extracts were probed with the indicated antibodies. (C) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole-cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells were cotransfected with PR55γ KD2 vector or control vector as indicated and incubated with PP2 for 2 h and UV for 1 h. Whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK) or JNK1 and JNK2 (α-JNK). (E) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of pSuper-c-SRC. Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), or SRC(α- SRC). (F) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of CDC42 V12 (dominant active). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 (α-JNK). (G) Cells were transfected with either the PP2A pool targeting PR55δ vector or control vector and cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). doi:10.1371/journal.pgen.0030218.g004

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Regulates JNK Upstream of MKK4 and at the Level or Upstream of c-SRC (A) pJNK and pMKK4 in relation to the total level of unphosphorylated protein in UV irradiated U2-OS cells followed over time (0–60 min) in the presence or absence of PR55γ KD2 vector. (B) U2-OS cells expressing PR55γ KD2 or a control vector were treated with UV and incubated for 60 min. Whole cell extracts were probed with the indicated antibodies. (C) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole-cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). (D) U2-OS cells were cotransfected with PR55γ KD2 vector or control vector as indicated and incubated with PP2 for 2 h and UV for 1 h. Whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK) or JNK1 and JNK2 (α-JNK). (E) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of pSuper-c-SRC. Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α- pJNK), JNK1 and JNK2 (α-JNK), or SRC(α- SRC). (F) PR55γ KD2 vector or control vector were cotransfected as indicated in the presence or absence of CDC42 V12 (dominant active). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 (α-JNK). (G) Cells were transfected with either the PP2A pool targeting PR55δ vector or control vector and cotransfected as indicated in the presence or absence of c-SRC 295M (dominant negative). Selected cells were exposed to UV irradiation and whole cell extracts were analyzed by immunoblotting with antibodies targeting phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK). doi:10.1371/journal.pgen.0030218.g004

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Irradiation, Plasmid Preparation, Expressing, Incubation, Dominant Negative Mutation, Transfection

    PP2A Family Screen (A) Schematic of the PP2A holoenzyme and outline of the B regulatory subunit families. (B) U2-OS cells were transfected with the indicated pSuper constructs and where available cotransfected with an HA-tagged version of the corresponding PP2A B subunit. Immunoblot panels show the efficiency of knockdown in six different pools as judged by the ability to knockdown cotransfected or endogenous protein (GFP is a transfection control). (C) U2-OS cells were cotransfected with pooled PP2A shRNAs or a control vector. Levels of phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK) were shown in cell lysates for the different samples 60 min after UV treatment of the cells. doi:10.1371/journal.pgen.0030218.g001

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PP2A Family Screen (A) Schematic of the PP2A holoenzyme and outline of the B regulatory subunit families. (B) U2-OS cells were transfected with the indicated pSuper constructs and where available cotransfected with an HA-tagged version of the corresponding PP2A B subunit. Immunoblot panels show the efficiency of knockdown in six different pools as judged by the ability to knockdown cotransfected or endogenous protein (GFP is a transfection control). (C) U2-OS cells were cotransfected with pooled PP2A shRNAs or a control vector. Levels of phosphorylated JNK (α-pJNK) or JNK1 and JNK2 (α-JNK) were shown in cell lysates for the different samples 60 min after UV treatment of the cells. doi:10.1371/journal.pgen.0030218.g001

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Transfection, Construct, Plasmid Preparation

    PR55γ Regulates c-SRC-Induced JNK Activation (A) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pSUPER-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (B) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pcDNA-HA-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (C) U2-OS cells were transfected with FLAG-SRC or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (D) U2-OS cells were cotransfected with FLAG-SRC, pSUPER-PR55γ, or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (E) U2-OS cells were cotransfected with FLAG-SRC, pcDNA-HA-PR55γ, or a empty vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (F) U2-OS cells expressing pcDNA-HA-hairpins targeting PR55γ or a control vector, and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. (G) U2-OS cells expressing pcDNA-HA-PR55γ or a control vector and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. doi:10.1371/journal.pgen.0030218.g006

    Journal: PLoS Genetics

    Article Title: A RNA Interference Screen Identifies the Protein Phosphatase 2A Subunit PR55? as a Stress-Sensitive Inhibitor of c-SRC

    doi: 10.1371/journal.pgen.0030218

    Figure Lengend Snippet: PR55γ Regulates c-SRC-Induced JNK Activation (A) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pSUPER-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (B) U2-OS cells expressing a 5× AP1-luciferase construct (pGL2), pcDNA-HA-PR55γ, or pcDNA-c-SRC as indicated. Luciferase counts are shown relative to the control. (C) U2-OS cells were transfected with FLAG-SRC or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (D) U2-OS cells were cotransfected with FLAG-SRC, pSUPER-PR55γ, or a control vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (E) U2-OS cells were cotransfected with FLAG-SRC, pcDNA-HA-PR55γ, or a empty vector and treated with UV. Whole cell extracts were probed with the indicated antibodies. (F) U2-OS cells expressing pcDNA-HA-hairpins targeting PR55γ or a control vector, and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. (G) U2-OS cells expressing pcDNA-HA-PR55γ or a control vector and pcDNA-c-SRC were serum starved for 48 h and treated with UV irradiation for 30 min. Cells were lysed in ELB and equivalent amounts of protein were immunoprecipitated with a c-SRC specific antibody. c-SRC phosphorylation was detected with an antibody targeting phosphorylated tyrosine 416 (α-pTyr 416 ) and immunoprecipitated c-SRC was detected with an α-c-SRC antibody. doi:10.1371/journal.pgen.0030218.g006

    Article Snippet: Antibodies anti-p-JNK, anti-p-MKK-4, anti-p-Src(416), and cleaved caspase-3 were from Cell Signaling; anti-SRC, anti-JNK (C-17), anti-MKK4, HA (Y11), anti-GFP, and anti-FYN were purchased from Santa Cruz Biotechnology Inc.

    Techniques: Activation Assay, Expressing, Luciferase, Construct, Transfection, Plasmid Preparation, Irradiation, Immunoprecipitation

    Up-regulated HRAS increased phosphorylated ERK1/2, JNK, and HSP27 and promoted cell motility and invasiveness. A , scrambled control siRNA oligonucleotides ( Scr )-transfected S2-013 cells and siRNA oligonucleotides targeting HRAS ( siHRAS )-transfected S2-013 cells were incubated on fibronectin. Western blotting ( IB ) was performed using the indicated antibodies. B , a Myc-tagged HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with scrambled control siRNA or HOXB7 siRNA with or without HRAS siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies. C , transwell motility and invasion assays. Oligonucleotides (siRNAs targeting HRAS or scrambled siRNAs as the negative control) were transiently transfected into S2-013 cells; 48 h later, transwell motility and Matrigel invasion assays were performed. Migrating cells in four visual fields per group were scored. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The transcription factor HOXB7 regulates ERK kinase activity and thereby stimulates the motility and invasiveness of pancreatic cancer cells

    doi: 10.1074/jbc.M116.772780

    Figure Lengend Snippet: Up-regulated HRAS increased phosphorylated ERK1/2, JNK, and HSP27 and promoted cell motility and invasiveness. A , scrambled control siRNA oligonucleotides ( Scr )-transfected S2-013 cells and siRNA oligonucleotides targeting HRAS ( siHRAS )-transfected S2-013 cells were incubated on fibronectin. Western blotting ( IB ) was performed using the indicated antibodies. B , a Myc-tagged HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with scrambled control siRNA or HOXB7 siRNA with or without HRAS siRNA; 48 h later, the cells were incubated on fibronectin. Western blotting was performed using the indicated antibodies. C , transwell motility and invasion assays. Oligonucleotides (siRNAs targeting HRAS or scrambled siRNAs as the negative control) were transiently transfected into S2-013 cells; 48 h later, transwell motility and Matrigel invasion assays were performed. Migrating cells in four visual fields per group were scored. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p

    Article Snippet: Anti-RhoA (sc-418), anti-MYC proto-oncogene (sc-789), anti-JNK (sc-7345), anti-phosphorylated JNK (sc-6254), anti-HSP27 (sc-13132), and anti-phosphorylated HSP27 (sc-166693) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-Rac1 (catalog no. 610650) and anti-Cdc42 (catalog no. 610929) antibodies were purchased from BD Transduction Laboratories (Palo Alto, CA).

    Techniques: Transfection, Incubation, Western Blot, Construct, Negative Control, Derivative Assay

    Association of HOXB7 with JNK and HSP27 in the motility and invasiveness of PDAC cells. A , a HOXB7-rescue construct was transfected into S2-013 cells that were transfected with scrambled control siRNA or HOXB7 siRNA; 48 h later, the cells were treated with or without SP600125 and assayed using motility and two-chamber invasion assays. Migrating cells in four fields per group were scored. Data are representative of three independent experiments. Columns , mean; bars , S.E. B , a HOXB7-rescue construct was transfected into scrambled control siRNA or HOXB7 siRNA-transfected S2-013 cells that had been transfected with or without HSP27 siRNA; 48 h later, motility and two-chamber invasion assays were performed. Migrating cells in four fields per group were counted. Data are derived from three independent experiments. Columns , mean; bars , S.E.

    Journal: The Journal of Biological Chemistry

    Article Title: The transcription factor HOXB7 regulates ERK kinase activity and thereby stimulates the motility and invasiveness of pancreatic cancer cells

    doi: 10.1074/jbc.M116.772780

    Figure Lengend Snippet: Association of HOXB7 with JNK and HSP27 in the motility and invasiveness of PDAC cells. A , a HOXB7-rescue construct was transfected into S2-013 cells that were transfected with scrambled control siRNA or HOXB7 siRNA; 48 h later, the cells were treated with or without SP600125 and assayed using motility and two-chamber invasion assays. Migrating cells in four fields per group were scored. Data are representative of three independent experiments. Columns , mean; bars , S.E. B , a HOXB7-rescue construct was transfected into scrambled control siRNA or HOXB7 siRNA-transfected S2-013 cells that had been transfected with or without HSP27 siRNA; 48 h later, motility and two-chamber invasion assays were performed. Migrating cells in four fields per group were counted. Data are derived from three independent experiments. Columns , mean; bars , S.E.

    Article Snippet: Anti-RhoA (sc-418), anti-MYC proto-oncogene (sc-789), anti-JNK (sc-7345), anti-phosphorylated JNK (sc-6254), anti-HSP27 (sc-13132), and anti-phosphorylated HSP27 (sc-166693) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-Rac1 (catalog no. 610650) and anti-Cdc42 (catalog no. 610929) antibodies were purchased from BD Transduction Laboratories (Palo Alto, CA).

    Techniques: Construct, Transfection, Derivative Assay

    Signaling pathway molecules associated with HOXB7 and ERK1/2. A , a HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with HOXB7 siRNA; 48 h later, the cells were incubated on fibronectin with or without U0126. Cell extracts were probed using human phosphoprotein arrays. B , densitometric analysis of the results of A . The levels of phosphorylated JNK and phosphorylated HSP27 in cells treated with U0126 were compared with those in the non-treated cells. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: The transcription factor HOXB7 regulates ERK kinase activity and thereby stimulates the motility and invasiveness of pancreatic cancer cells

    doi: 10.1074/jbc.M116.772780

    Figure Lengend Snippet: Signaling pathway molecules associated with HOXB7 and ERK1/2. A , a HOXB7-rescue construct was transfected into S2-013 cells that had been transfected with HOXB7 siRNA; 48 h later, the cells were incubated on fibronectin with or without U0126. Cell extracts were probed using human phosphoprotein arrays. B , densitometric analysis of the results of A . The levels of phosphorylated JNK and phosphorylated HSP27 in cells treated with U0126 were compared with those in the non-treated cells. Data are derived from three independent experiments. Columns , mean; bars , S.E. *, p

    Article Snippet: Anti-RhoA (sc-418), anti-MYC proto-oncogene (sc-789), anti-JNK (sc-7345), anti-phosphorylated JNK (sc-6254), anti-HSP27 (sc-13132), and anti-phosphorylated HSP27 (sc-166693) antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-Rac1 (catalog no. 610650) and anti-Cdc42 (catalog no. 610929) antibodies were purchased from BD Transduction Laboratories (Palo Alto, CA).

    Techniques: Construct, Transfection, Incubation, Derivative Assay

    Neuroprotection in hippocampal CA3 neurons, and the prevention of p-JNK, BACE1 and NOX2 induction by early continuous 17β-oestradiol (E2) replacement for 10 weeks in rats with LTED. ( A ) Representative staining of NeuN, TUNEL and amyloid-β1–42 of CA3 region from sham (Sh), and animals treated with 17β-oestradiol or placebo (Pla) immediately after ovariectomy for the entire 10-week period. The rats were subjected to sham or 10 min ischaemia followed by 7 days of reperfusion. ( B ) Cell-counting study shows the number of surviving neurons (% changes of sham) and TUNEL-positive cells per 250 µm length of medial CA3 region. Magnification: ×40. Scale bar = 50 µm. n = 6–7 in each group. # P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Neuroprotection in hippocampal CA3 neurons, and the prevention of p-JNK, BACE1 and NOX2 induction by early continuous 17β-oestradiol (E2) replacement for 10 weeks in rats with LTED. ( A ) Representative staining of NeuN, TUNEL and amyloid-β1–42 of CA3 region from sham (Sh), and animals treated with 17β-oestradiol or placebo (Pla) immediately after ovariectomy for the entire 10-week period. The rats were subjected to sham or 10 min ischaemia followed by 7 days of reperfusion. ( B ) Cell-counting study shows the number of surviving neurons (% changes of sham) and TUNEL-positive cells per 250 µm length of medial CA3 region. Magnification: ×40. Scale bar = 50 µm. n = 6–7 in each group. # P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Staining, TUNEL Assay, Proximity Ligation Assay, Cell Counting

    Activation and role of NOX2 NADPH oxidase and JNK/cJun signalling in CA3 neuronal cell death following LTED plus cerebral ischaemia. ( A ) Western blot analyses of NOX2 protein from CA3 samples at 3 h reperfusion in short/long term rats. ( B ) Representative microscopy images of NeuN (red) and NOX2 (green) in CA3 regions of placebo groups at 3 h ischaemic reperfusion. Magnification: ×40. Scale bar = 50 µm. ( C ) Changes of NADPH oxidase activity and superoxide production in CA3 region at 3 h reperfusion in the LTED animals. ( D and E ) Western blots of CA3 protein samples show activation of JNK and cJun induced by 3 h ischaemic reperfusion and the inhibition by the competitive NOX2 inhibitor gp91ds-tat peptide (91ds-tat) but not scrambled control peptide (Scr). ( F ) Administration of the JNK inhibitor SP600125 (SP) or 91ds-tat prevents cJun/AP-1 activity in CA3 samples at 3 h reperfusion in the LTED groups. ( G ) Quantitative data ( n = 5–7/group) showing the neuroprotective effects of 91ds-tat and SP in hippocampal CA3 region of rats with LTED at Day 7 of ischaemic reperfusion. A–F : n = 4–5 per group. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Activation and role of NOX2 NADPH oxidase and JNK/cJun signalling in CA3 neuronal cell death following LTED plus cerebral ischaemia. ( A ) Western blot analyses of NOX2 protein from CA3 samples at 3 h reperfusion in short/long term rats. ( B ) Representative microscopy images of NeuN (red) and NOX2 (green) in CA3 regions of placebo groups at 3 h ischaemic reperfusion. Magnification: ×40. Scale bar = 50 µm. ( C ) Changes of NADPH oxidase activity and superoxide production in CA3 region at 3 h reperfusion in the LTED animals. ( D and E ) Western blots of CA3 protein samples show activation of JNK and cJun induced by 3 h ischaemic reperfusion and the inhibition by the competitive NOX2 inhibitor gp91ds-tat peptide (91ds-tat) but not scrambled control peptide (Scr). ( F ) Administration of the JNK inhibitor SP600125 (SP) or 91ds-tat prevents cJun/AP-1 activity in CA3 samples at 3 h reperfusion in the LTED groups. ( G ) Quantitative data ( n = 5–7/group) showing the neuroprotective effects of 91ds-tat and SP in hippocampal CA3 region of rats with LTED at Day 7 of ischaemic reperfusion. A–F : n = 4–5 per group. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Microscopy, Activity Assay, Inhibition

    Activation of JNK pathway induces amyloid precursor protein (T668) phosphorylation and an increase of C-99/C-83 amyloid precursor protein ratio in rats with LTED, and hypersensitivity of LTED CA3 neurons extends to the Alzheimer’s disease-relevant insult, amyloid-β1–42. ( A ) Representative confocal analysis shows increased immunoreactivity of p-APP (T668) and p-JNK in LTED CA3 neurons. ( B ) Western blot analyses of p-APP (T668) and total amyloid precursor protein in CA3 protein samples. ( C ) Western blot analysis specific for C-terminal fragment of amyloid precursor protein after α-secretase cleavage (C-83) and β-secretase cleavage (C-99). Ratios of C-99 fragments to C-83 fragments were expressed as fold changes versus the value in the short-term sham. JNK inhibition provoked a significant reduction of the C-99/C-83 ratio, indicating a shift towards the non-amyloidogenic pathway. ( D ) ELISA assay of amyloid-β1–42 and amyloid-β1–40 was performed by using hippocampal CA3 homogenates at 3 h ischaemic reperfusion ( a , b ). ( E ) Typical microscopy images showing the double staining of amyloid-β1–42 and TUNEL in CA3 region at 7 days reperfusion. Note that amyloid-β1–42 significantly merged with TUNEL-positive apoptotic-like CA3 neurons in rats with LTED. ( F ) Hypersensitivity of hippocampal CA3 neurons to amyloid-β1–42-induced neurotoxicity in rats with LTED. ( a ) Typical staining of NeuN in short/long-term 17β-oestradiol (E2) deprived rats at day 7 after infusion with amyloid-β1–42 or scrambled amyloid-β1–42 into the CA3 region of the hippocampus. ( b ) Quantitative analysis of data shows the numbers of surviving neurons in CA3 region. Scale bar = 50 µm. Magnification: ×40. A–E : n = 4–6 per group. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Activation of JNK pathway induces amyloid precursor protein (T668) phosphorylation and an increase of C-99/C-83 amyloid precursor protein ratio in rats with LTED, and hypersensitivity of LTED CA3 neurons extends to the Alzheimer’s disease-relevant insult, amyloid-β1–42. ( A ) Representative confocal analysis shows increased immunoreactivity of p-APP (T668) and p-JNK in LTED CA3 neurons. ( B ) Western blot analyses of p-APP (T668) and total amyloid precursor protein in CA3 protein samples. ( C ) Western blot analysis specific for C-terminal fragment of amyloid precursor protein after α-secretase cleavage (C-83) and β-secretase cleavage (C-99). Ratios of C-99 fragments to C-83 fragments were expressed as fold changes versus the value in the short-term sham. JNK inhibition provoked a significant reduction of the C-99/C-83 ratio, indicating a shift towards the non-amyloidogenic pathway. ( D ) ELISA assay of amyloid-β1–42 and amyloid-β1–40 was performed by using hippocampal CA3 homogenates at 3 h ischaemic reperfusion ( a , b ). ( E ) Typical microscopy images showing the double staining of amyloid-β1–42 and TUNEL in CA3 region at 7 days reperfusion. Note that amyloid-β1–42 significantly merged with TUNEL-positive apoptotic-like CA3 neurons in rats with LTED. ( F ) Hypersensitivity of hippocampal CA3 neurons to amyloid-β1–42-induced neurotoxicity in rats with LTED. ( a ) Typical staining of NeuN in short/long-term 17β-oestradiol (E2) deprived rats at day 7 after infusion with amyloid-β1–42 or scrambled amyloid-β1–42 into the CA3 region of the hippocampus. ( b ) Quantitative analysis of data shows the numbers of surviving neurons in CA3 region. Scale bar = 50 µm. Magnification: ×40. A–E : n = 4–6 per group. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Inhibition, Enzyme-linked Immunosorbent Assay, Microscopy, Double Staining, TUNEL Assay, Staining

    BACE1 protein induction is mediated by activation of JNK/cJun signalling following ischaemic reperfusion in rats with LTED. ( A ) Western blot analyses of BACE1 and β-actin were performed with hippocampal CA3 protein in the indicated groups. BACE1 protein elevation induced by 3 h ischaemic reperfusion in rats with LTED was decreased by the JNK inhibitor SP600125 (SP). ( B ) Representative confocal analysis demonstrates increased immunofluorescence of BACE1 expression and active JNK/cJun in CA3 neurons in rats with LTED at 3 h reperfusion. Note that increased p-JNK/p-cJun staining co-localized well in the same cells with BACE1 protein in rats with LTED. ( C ) BACE1 activity in the indicated CA3 homogenates was assessed by measuring the liberated fluorescent fragment based on the BACE1-cleavable peptide substrate. ( D and E ) Representative sections of in situ hybridization for messenger RNA expression of BACE1 from sham ( a ), placebo ( b ), vehicle ( c ) and SP600125 ( d ) treated rats with LTED. Quantitative analysis of BACE1 messenger RNA expression in the CA3 pyramidal cell layer. Data are expressed as means ± SE from four to five rats per group, and shown as fold-change or percent-change versus the value in the short-term sham. * P

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: BACE1 protein induction is mediated by activation of JNK/cJun signalling following ischaemic reperfusion in rats with LTED. ( A ) Western blot analyses of BACE1 and β-actin were performed with hippocampal CA3 protein in the indicated groups. BACE1 protein elevation induced by 3 h ischaemic reperfusion in rats with LTED was decreased by the JNK inhibitor SP600125 (SP). ( B ) Representative confocal analysis demonstrates increased immunofluorescence of BACE1 expression and active JNK/cJun in CA3 neurons in rats with LTED at 3 h reperfusion. Note that increased p-JNK/p-cJun staining co-localized well in the same cells with BACE1 protein in rats with LTED. ( C ) BACE1 activity in the indicated CA3 homogenates was assessed by measuring the liberated fluorescent fragment based on the BACE1-cleavable peptide substrate. ( D and E ) Representative sections of in situ hybridization for messenger RNA expression of BACE1 from sham ( a ), placebo ( b ), vehicle ( c ) and SP600125 ( d ) treated rats with LTED. Quantitative analysis of BACE1 messenger RNA expression in the CA3 pyramidal cell layer. Data are expressed as means ± SE from four to five rats per group, and shown as fold-change or percent-change versus the value in the short-term sham. * P

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activation Assay, Western Blot, Immunofluorescence, Expressing, Staining, Activity Assay, In Situ Hybridization, RNA Expression

    Summary diagram of the proposed mechanisms underlying CA3 hypersensitivity, Alzheimer’s disease protein induction, neuronal death and behavioural deficits following cerebral ischaemia in rats with LTED. LTED plus stress (cerebral ischaemia) leads to hyperinduction of NOX2 NADPH oxidase activity and superoxide elevation, resulting in the activation of stress-responsive JNK/cJun signalling pathway in hippocampal CA3 neurons. JNK pathway activation induces the gene expression of BACE1 as well as amyloid precursor protein (Thr668) phosphorylation, which together promotes amyloid-β expression following ischaemic stress. JNK/cJun activation also initiates a positive-feedback regulation of NADPH oxidase and superoxide production by inducing NOX2 expression. Finally, Tau hyperphosphorylation resulting from JNK activation, together with amyloid-β generation and other possible factors induced by JNK/cJun signalling, leads to enhanced CA3 neuronal cell death and memory deterioration following cerebral ischaemia in rats with LTED. APP = amyloid precursor protein.

    Journal: Brain

    Article Title: Hypersensitivity of the hippocampal CA3 region to stress-induced neurodegeneration and amyloidogenesis in a rat model of surgical menopause

    doi: 10.1093/brain/awt046

    Figure Lengend Snippet: Summary diagram of the proposed mechanisms underlying CA3 hypersensitivity, Alzheimer’s disease protein induction, neuronal death and behavioural deficits following cerebral ischaemia in rats with LTED. LTED plus stress (cerebral ischaemia) leads to hyperinduction of NOX2 NADPH oxidase activity and superoxide elevation, resulting in the activation of stress-responsive JNK/cJun signalling pathway in hippocampal CA3 neurons. JNK pathway activation induces the gene expression of BACE1 as well as amyloid precursor protein (Thr668) phosphorylation, which together promotes amyloid-β expression following ischaemic stress. JNK/cJun activation also initiates a positive-feedback regulation of NADPH oxidase and superoxide production by inducing NOX2 expression. Finally, Tau hyperphosphorylation resulting from JNK activation, together with amyloid-β generation and other possible factors induced by JNK/cJun signalling, leads to enhanced CA3 neuronal cell death and memory deterioration following cerebral ischaemia in rats with LTED. APP = amyloid precursor protein.

    Article Snippet: The following primary antibodies were used in different combinations: anti-NeuN (1:500, MAB377, Millipore); anti-amyloid-β[1-42] (1:500, #700254) and anti-BACE1 (1:200, AHB0241) from Invitrogen Corporation; anti-phospho-JNK (1:50, sc-12882) and anti-phospho-cJun (1:50, sc-7981) from Santa Cruz Biotechnology, anti-phospho-amyloid precursor protein (1:400, #3823, Cell Signaling Technology), anti-PHF1 (1:2000; gift from Dr. Peter Davies).

    Techniques: Activity Assay, Activation Assay, Expressing

    Structure of JNK1 in complex with MKP7-CD. ( a ) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. ( b ) Structure of MKP7-CD with its active site highlight in cyan. The 2 F o − F c omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b . ( c ) Structure of VHR with its active site highlighted in marine blue. ( d ) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). ( e ) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). Blue dashed lines represent polar interactions. ( f ) The 2 F o − F c omit map (contoured at 1.5σ) clearly shows electron density for the 285 FNFL 288 segment of MKP7-CD.

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: Structure of JNK1 in complex with MKP7-CD. ( a ) Ribbon diagram of JNK1–MKP7-CD complex in two views related by a 45° rotation around a vertical axis. ( b ) Structure of MKP7-CD with its active site highlight in cyan. The 2 F o − F c omit map (contoured at 1.5σ) for the P-loop of MKP7-CD is shown at inset of b . ( c ) Structure of VHR with its active site highlighted in marine blue. ( d ) Close-up view of the JNK1–MKP7 interface showing interacting amino acids of JNK1 (orange) and MKP7-CD (cyan). The JNK1 is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). ( e ) Interaction networks mainly involving helices α4 and α5 from MKP7-CD, and αG and α2L14 of JNK1. MKP7-CD is shown in surface representation coloured according to electrostatic potential (positive, blue; negative, red). Blue dashed lines represent polar interactions. ( f ) The 2 F o − F c omit map (contoured at 1.5σ) clearly shows electron density for the 285 FNFL 288 segment of MKP7-CD.

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques:

    MKP7-CD is crucial for JNK1 binding and enzyme catalysis. ( a ) Domain organization of human MKP7 and JNK1. The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. The key structural elements are indicated. The colour scheme is the same in the following figures unless indicated otherwise. ( b ) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to equation (1) . Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. ( c ) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. ( d ) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. The protein amounts of MKP7 used are shown at the bottom.

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: MKP7-CD is crucial for JNK1 binding and enzyme catalysis. ( a ) Domain organization of human MKP7 and JNK1. The KBD and CD of MKP7 are shown in green and blue, and the N-lobe and C-lobe of JNK1 are coloured in lemon and yellow, respectively. The key structural elements are indicated. The colour scheme is the same in the following figures unless indicated otherwise. ( b ) Plots of initial velocity of the MKP7-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to equation (1) . Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. ( c ) Gel filtration analysis for interaction of JNK1 with MKP7-CD and MKP7-KBD. ( d ) GST-mediated pull-down assay for interaction of JNK1 with MKP7-CD and MKP7-KBD. The top panel shows the relative affinities of MKP7-CD and MKP7-KBD to JNK1, with the affinity of MKP7-CD defined as 100%; the middle panel is the electrophoretic pattern of MKP7 and JNK1 after GST pull-down assays. The protein amounts of MKP7 used are shown at the bottom.

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques: Binding Assay, Concentration Assay, Filtration, Pull Down Assay

    Comparison of CDK2-KAP and JNK1–MKP7-CD. ( a ) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). ( b ) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. The orientation of the panel is almost identical to those of Fig. 3d . The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red). Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. Blue dashed lines represent polar interactions. One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. ( c ) Sequence alignment of the JNK-interacting regions on MKPs. Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. ( d ) Sequence alignment of the F-site regions on MAPKs. Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow.

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: Comparison of CDK2-KAP and JNK1–MKP7-CD. ( a ) Superposition of the complex structures of CDK2-KAP (PDB 1FQ1) and JNK1–MKP7-CD. The N-lobe and C-lobe of CDK2 are coloured in grey and pink, respectively, and KAP is coloured in green. The interactions between these two proteins consist of three discontinuous contact regions, centred at the multiple hydrogen bonds between the pThr160 of CDK2 and the active site of KAP (region I). Interestingly, the recognition of CDK2 by KAP is augmented by a similar interface as that observed in the complex of JNK1 and MKP7-CD (region II). ( b ) Interactions networks at the auxiliary region II mainly involving helix α7 from KAP and the αG helix and following L14 loop of CDK2. The orientation of the panel is almost identical to those of Fig. 3d . The CDK2 is shown in surface representation coloured according to the electrostatic potential (positive, blue; negative, red). Residues of KAP and CDK2 are highlighted as green and red sticks, respectively. Blue dashed lines represent polar interactions. One remarkable difference between these two kinase-phosphatase complexes is that helix α6 of KAP (corresponding to helix α4 of MKP7-CD) plays little, if any, role in the formation of a stable heterodimer of CDK2 and KAP. ( c ) Sequence alignment of the JNK-interacting regions on MKPs. Residues of MKP7-CD involved in JNK1 recognition are indicated by cyan asterisks, and the conserved FXF-motif is highlighted in cyan. The secondary structure assignments of MKP7-CD and KAP are shown above and below each sequence. ( d ) Sequence alignment of the F-site regions on MAPKs. Residues of JNK1 involved in recognition of MKP7 are indicated by orange asterisks, and those forming the F-site are highlighted in yellow.

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques: Sequencing

    Mutational analysis on interactions between MKP7-CD and JNK1. ( a ) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n =3). Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. ( b ) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. ( c ) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. The protein amounts of MKP7-CD used are shown at the bottom. ( d ) Circular dichroism spectra for MKP7-CD wild type and mutants. Measurements were averaged for three scans. ( e ) Circular dichroism spectra for JNK1 wild type and mutants. Measurements were averaged for three scans. ( f ) Effects of mutations in MKP7-CD on the p NPP hydrolysis reaction (mean±s.e.m., n =3).

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: Mutational analysis on interactions between MKP7-CD and JNK1. ( a ) Effects of mutations in MKP7-CD on the JNK1 dephosphorylation (mean±s.e.m., n =3). Residues involved in hydrophobic and hydrophilic contacts are coloured in red and blue, respectively. ( b ) Gel filtration analysis for interaction of JNK1 with MKP7-CD mutant F285D. Mutant F285D and JNK1 were eluted as monomers, with the molecular masses of ∼17 and 44 kDa, respectively. However, in contrast to the wild-type MKP7-CD, mutant F285D did not co-migrate with JNK1. ( c ) Pull-down assays of MKP7-CD by GST-tagged JNK1 mutants. The top panel shows the relative affinities of MKP7-CD to JNK1 mutants, with the affinity of wild-type JNK1 defined as 100%, the middle panel is the electrophoretic pattern of MKP7-CD and JNK1 mutants after GST pull-down assays. The protein amounts of MKP7-CD used are shown at the bottom. ( d ) Circular dichroism spectra for MKP7-CD wild type and mutants. Measurements were averaged for three scans. ( e ) Circular dichroism spectra for JNK1 wild type and mutants. Measurements were averaged for three scans. ( f ) Effects of mutations in MKP7-CD on the p NPP hydrolysis reaction (mean±s.e.m., n =3).

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques: De-Phosphorylation Assay, Filtration, Mutagenesis

    FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. ( a – c ) FXF-motif is essential for the dephosphorylation of JNK by MKP7. HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). After 36 h infection, cells were untreated in a , stimulated with 30 μM etoposide for 3 h in b or irradiated with 25 J m −2 ultraviolet light at 30 min before lysis in c . Whole-cell extracts were then immunoblotted with antibody indicated. Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. ( d ) F-site is required for JNK1 to interact with MKP7. HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). At 16 h post transfection, cells were lysed. Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. IP, immunoprecipitation; TCL, total cell lysate. Shown is a typical result from three independent experiments. ( e ) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. At 36 h post infection, cells were irradiated with 25 J m −2 ultraviolet light and collected at 6 h after irradiation. Cells were then subjected to flow cytometry analysis. Apoptotic cells were determined by Annexin-V-APC/PI staining. The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). The values shown in the lower left, and upper right quadrants of each panel represent the percentage of viable, and apoptotic cells, respectively. All results are representative of three independent experiments. ( f ) Statistical analysis of apoptotic cells (mean±s.e.m., n =3), * P

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: FXF-motif is critical for controlling the phosphorylation of JNK and ultraviolet-induced apoptosis. ( a – c ) FXF-motif is essential for the dephosphorylation of JNK by MKP7. HEK293T cells were infected with lentiviruses expressing MKP7 and its mutants (1.0 μg). After 36 h infection, cells were untreated in a , stimulated with 30 μM etoposide for 3 h in b or irradiated with 25 J m −2 ultraviolet light at 30 min before lysis in c . Whole-cell extracts were then immunoblotted with antibody indicated. Shown is a typical immunoblot for phosphorylated JNK from three independent experiments. ( d ) F-site is required for JNK1 to interact with MKP7. HEK293T cells were co-transfected with MKP7 full-length (1.0 μg) and JNK1 (wild type or mutants as indicated, 1.0 μg). At 16 h post transfection, cells were lysed. Whole-cell extracts were then immunoprecipitated with antibody against Myc for MKP7; immunobloting was carried out with antibodies indicated. IP, immunoprecipitation; TCL, total cell lysate. Shown is a typical result from three independent experiments. ( e ) Effect of MKP7 (wild type or mutants) expression on ultraviolet-induced apoptosis. HeLa cells were infected with lentiviruses expressing MKP7 full-length and its mutants. At 36 h post infection, cells were irradiated with 25 J m −2 ultraviolet light and collected at 6 h after irradiation. Cells were then subjected to flow cytometry analysis. Apoptotic cells were determined by Annexin-V-APC/PI staining. The results using Annexin-V stain for membrane phosphatidylserine eversion, combined with propidium iodide (PI) uptake to evaluate cells whose membranes had been compromised. Staining with both Annexin-V and PI indicate apoptosis (upper right quadrant). The values shown in the lower left, and upper right quadrants of each panel represent the percentage of viable, and apoptotic cells, respectively. All results are representative of three independent experiments. ( f ) Statistical analysis of apoptotic cells (mean±s.e.m., n =3), * P

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques: De-Phosphorylation Assay, Infection, Expressing, Irradiation, Lysis, Transfection, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Staining

    MKP5-CD is crucial for JNK1 binding and enzyme catalysis. ( a ) Domain organization of human MKP5. The KBD and CD of MKP5 are shown in brown and grey, respectively. ( b ) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to the Michaelis–Menten equation with K m and k cat values indicated. Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. ( c ) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. ( d ) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. ( e ) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. The panels are arranged the same as in Fig. 2d . ( f ) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n =3). ( g ) Effects of mutations in MKP5-CD on the p NPP hydrolysis reaction (mean±s.e.m., n =3). ( h ) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. The panels are arranged the same as in Fig. 4c .

    Journal: Nature Communications

    Article Title: A conserved motif in JNK/p38-specific MAPK phosphatases as a determinant for JNK1 recognition and inactivation

    doi: 10.1038/ncomms10879

    Figure Lengend Snippet: MKP5-CD is crucial for JNK1 binding and enzyme catalysis. ( a ) Domain organization of human MKP5. The KBD and CD of MKP5 are shown in brown and grey, respectively. ( b ) Plots of initial velocity of the MKP5-catalysed reaction versus phospho-JNK1 concentration. The solid lines are best-fitting results according to the Michaelis–Menten equation with K m and k cat values indicated. Each experiment was performed in replicate for at least three times. The error bars represent s.e.m. ( c ) Structural comparison of the JNK-interacting residues on MKP5-CD (PDB 1ZZW) and MKP7-CD. The corresponding residues on MKP5 are depicted as orange sticks, and MKP5 residues numbers are in parentheses. ( d ) Gel filtration analysis for interaction of JNK1 with MKP5-CD and MKP5-KBD. ( e ) GST-mediated pull-down assays for interaction of JNK1 with MKP5-CD and MKP5-KBD. The panels are arranged the same as in Fig. 2d . ( f ) Effects of mutations in MKP5-CD on the JNK1 dephosphorylation (mean±s.e.m., n =3). ( g ) Effects of mutations in MKP5-CD on the p NPP hydrolysis reaction (mean±s.e.m., n =3). ( h ) Pull-down assays of MKP5-CD by GST-tagged JNK1 mutants. The panels are arranged the same as in Fig. 4c .

    Article Snippet: Antibodies and drugs Antibodies used in this study: mouse anti-HA (1:100 for immunoprecipitation; F-7) and anti-JNK1 (1:1,000 for immunoblotting; F-3) antibodies were purchased from Santa-Cruz Biotechnology.

    Techniques: Binding Assay, Concentration Assay, Filtration, De-Phosphorylation Assay

    Resveratrol inhibits S. aureus -induced VCAM-1 expression and inflammatory signaling pathways. ( A ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. ( B ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 24 h. The THP-1 cells adherence was measured. ( C – E ) Cells were pretreated without or with resveratrol for 24 h, and then incubated with S. aureus for the indicated times. The expression of phospho-c-Src, phospho-PDGFR, phospho-p38 MAPK, phospho-JNK1/2, phospho-c-Jun, and phospho-ATF2 were determined by Western blot. ( F ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h. ATF2 and c-Jun binding activities were analyzed by a ChIP assay. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: Resveratrol inhibits S. aureus -induced VCAM-1 expression and inflammatory signaling pathways. ( A ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h or 6 h. The mRNA levels and promoter activity of VCAM-1 were determined. ( B ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 24 h. The THP-1 cells adherence was measured. ( C – E ) Cells were pretreated without or with resveratrol for 24 h, and then incubated with S. aureus for the indicated times. The expression of phospho-c-Src, phospho-PDGFR, phospho-p38 MAPK, phospho-JNK1/2, phospho-c-Jun, and phospho-ATF2 were determined by Western blot. ( F ) Cells were pretreated with resveratrol for 24 h, and then incubated with S. aureus for 4 h. ATF2 and c-Jun binding activities were analyzed by a ChIP assay. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Incubation, Activity Assay, Western Blot, Binding Assay, Chromatin Immunoprecipitation

    S. aureus induces AP-1 activation via c-Src/PDGFR/JNK1/2 and p38 MAPK pathways. Cells were pretreated without or with PP1, AG1296, SP600125, or SB202190 for 1 h, and then incubated with S. aureus for the indicated times. The expression of ( A ) phospho-c-Jun and ( B ) phospho-ATF2 was determined by Western blot. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: S. aureus induces AP-1 activation via c-Src/PDGFR/JNK1/2 and p38 MAPK pathways. Cells were pretreated without or with PP1, AG1296, SP600125, or SB202190 for 1 h, and then incubated with S. aureus for the indicated times. The expression of ( A ) phospho-c-Jun and ( B ) phospho-ATF2 was determined by Western blot. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Activation Assay, Incubation, Expressing, Western Blot

    Schematic diagram illustrating the proposed signaling pathway involved in S. aureus -induced VCAM-1 expression in HPAEpiCs. S. aureus induces JNK1/2 and p38 MAPK activation via a c-Src/PDGFR pathway, which in turn initiates the activation of AP-1. Activated AP-1 is recruited to the promoter regions of VCAM-1, leading to an increase of VCAM-1 promoter activity and the expression of VCAM-1 mRNA and protein in HPAEpiCs. Moreover, resveratrol can reduce lung inflammation via the inhibition of VCAM-1 expression, monocyte adhesion, and the activation of c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 induced by S. aureus .

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: Schematic diagram illustrating the proposed signaling pathway involved in S. aureus -induced VCAM-1 expression in HPAEpiCs. S. aureus induces JNK1/2 and p38 MAPK activation via a c-Src/PDGFR pathway, which in turn initiates the activation of AP-1. Activated AP-1 is recruited to the promoter regions of VCAM-1, leading to an increase of VCAM-1 promoter activity and the expression of VCAM-1 mRNA and protein in HPAEpiCs. Moreover, resveratrol can reduce lung inflammation via the inhibition of VCAM-1 expression, monocyte adhesion, and the activation of c-Src, PDGFR, JNK1/2, p38 MAPK, and AP-1 induced by S. aureus .

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Activation Assay, Activity Assay, Inhibition

    S. aureus induces VCAM-1 expression in HPAEpiCs via PDGFR. ( A ) Cells were transfected with scrambled, p42, p38, or JNK1 siRNA, and then incubated with S. aureus for 4 h, 6 h, or 24 h. The mRNA levels and promoter activity of VCAM-1 and the THP-1 cells adherence were measured. ( B ) Cells were pretreated without or with PP1 or AG1296 for 1 h, and then incubated with S. aureus for the indicated times. The expression of phospho-p38 MAPK, phospho-p42/p44 MAPK, and phospho-JNK1/2 were determined by Western blot. n = 3–4, # p

    Journal: International Journal of Molecular Sciences

    Article Title: Resveratrol Attenuates Staphylococcus Aureus-Induced Monocyte Adhesion through Downregulating PDGFR/AP-1 Activation in Human Lung Epithelial Cells

    doi: 10.3390/ijms19103058

    Figure Lengend Snippet: S. aureus induces VCAM-1 expression in HPAEpiCs via PDGFR. ( A ) Cells were transfected with scrambled, p42, p38, or JNK1 siRNA, and then incubated with S. aureus for 4 h, 6 h, or 24 h. The mRNA levels and promoter activity of VCAM-1 and the THP-1 cells adherence were measured. ( B ) Cells were pretreated without or with PP1 or AG1296 for 1 h, and then incubated with S. aureus for the indicated times. The expression of phospho-p38 MAPK, phospho-p42/p44 MAPK, and phospho-JNK1/2 were determined by Western blot. n = 3–4, # p

    Article Snippet: Anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-phospho-p42/p44 MAPK, anti-phospho-c-Src, anti-phospho-PDGFR, anti-phospho-ATF2, and anti-phospho-c-Jun antibodies were obtained from Cell Signaling (Danver, MA, USA).

    Techniques: Expressing, Transfection, Incubation, Activity Assay, Western Blot

    IL-1β induces linker phosphorylation of Smad3 in HSCs. (A) HSCs-P1 were treated with R-III (0.3 μM) for 20 h and cell lysates were analyzed by western blotting. The Western blots are representative of three independent experiments from separate cell preparations. α-tubulin was used as a loading control. (B) HSCs-P1 were treated with R-III in the presence of IL-1RA (1 μg/ml) and analyzed by western blotting. (C, D) HSCs-P1 were treated with R-III in the presence of the inhibitor for TAK1 ((5Z)-7-Oxozeaenol, 0.25 μM), p38 (SB203580, 5 μM) and JNK (SP600125, 10 μM) and analyzed by western blotting (C) and real-time PCR (D). P -value, paired t-test (n = 3) (compared to untreated HSCs-P1), *P

    Journal: bioRxiv

    Article Title: Albumin inhibits the activation of hepatic stellate cells by suppressing TGF-β/Smad3 signaling via IL-1β

    doi: 10.1101/753152

    Figure Lengend Snippet: IL-1β induces linker phosphorylation of Smad3 in HSCs. (A) HSCs-P1 were treated with R-III (0.3 μM) for 20 h and cell lysates were analyzed by western blotting. The Western blots are representative of three independent experiments from separate cell preparations. α-tubulin was used as a loading control. (B) HSCs-P1 were treated with R-III in the presence of IL-1RA (1 μg/ml) and analyzed by western blotting. (C, D) HSCs-P1 were treated with R-III in the presence of the inhibitor for TAK1 ((5Z)-7-Oxozeaenol, 0.25 μM), p38 (SB203580, 5 μM) and JNK (SP600125, 10 μM) and analyzed by western blotting (C) and real-time PCR (D). P -value, paired t-test (n = 3) (compared to untreated HSCs-P1), *P

    Article Snippet: Primary antibodies used were α-SMA (Sigma-Aldrich #A2547, St. Louis, MO, USA), Smad2, p-Smad2 (S465,467), Smad3, p-Smad3 (S423,425), TAK1, p-TAK1 (S412), p38, p-p38, JNK, p-JNK, α-tubulin (#5339, 3101, 9523, 9520, 4505, 9339, 9212, 9211, 9252, 9255, 2125; Cell Signaling Technology, Beverly, MA, USA), p-Smad3 (T179) (Merck #ABS47, Darmstadt, Germany), p-Smad3 (S208), p-TAK1 (T184,187) (#PA5-38521, MA5-15073; Thermo Fisher Scientific, Waltham, MA, USA), PPAR-γ, RARα (#ab41928, ab41934; Abcam, Cambridge, UK), RXR (Santa Cruz Biotechnology #sc-774, Dallas, TX, USA).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction