vector pmt3 p38  (Addgene inc)

Journal: Journal of cell science

Article Title: p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

doi: 10.1242/jcs.032854

Figure Lengend Snippet: Fig. 1. Stimulation of Frizzled-1 by Wnt3a activates p38 MAPK in F9 and HEK293 cells. (A) F9 cells stably expressing Rfz1 were treated with Wnt3a (20 ng/ml) for indicated periods of time and the lysates were assayed for p38 activation as described in Materials and Methods. The upper panel represents mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with either anti-ATF2-P (p- ATF2), upper panel or with anti-p38 antibody (p38), lower panel. (B) F9 cells alone and F9 cells expressing either Rfz2 or Rfz1 were treated with Wnt3a for 15 minutes and the lysates were assayed for p38 activation. Representative blots of three independent experiments that proved highly reproducible are shown. (C) Confluent HEK293 cells were treated with Wnt3a for 15 minutes and the lysates were assayed for p38 activation. Representative blots of three independent experiments that proved highly reproducible are displayed. *P<0.05; **P<0.01 versus control (+Wnt3a, 0 minutes).

Article Snippet: Rescue experiments were performed by transfecting F9 cells stably expressing Rfz1 and the pTOPFLASH (M50) luciferase reporter with an expression vector harboring siRNA-resistant hp38 (pMT3-p38-HA, Addgene plasmid 12658).

Techniques: Stable Transfection, Expressing, Activation Assay, Control

Journal: Journal of cell science

Article Title: p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

doi: 10.1242/jcs.032854

Figure Lengend Snippet: Fig. 2. Suppression of Gαq and Gαs, but not Gαo or Gα11, attenuates Wnt3a- induced p38 MAPK activation. F9 cells expressing Rfz1 were treated with siRNAs specific for Gαo, Gαq, Gα11 or Gαs for 48 hours before treatment with Wnt3a for 15 minutes. p38 MAPK activity was then assayed. Upper panel displays mean values ± s.e.m. obtained from densitometer scanning of images from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38), anti-Gαo(Gαo), anti- Gαq (Gαq), anti-Gα11(Gα11) and anti-Gαs (Gαs). *P<0.05 versus –Wnt3a control; #P<0.05 versus +Wnt3a control. The extent of knockdown of the G- protein α-subunit expression was routinely 75% or more.

Article Snippet: Rescue experiments were performed by transfecting F9 cells stably expressing Rfz1 and the pTOPFLASH (M50) luciferase reporter with an expression vector harboring siRNA-resistant hp38 (pMT3-p38-HA, Addgene plasmid 12658).

Techniques: Activation Assay, Expressing, Activity Assay, Control, Knockdown

Journal: Journal of cell science

Article Title: p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

doi: 10.1242/jcs.032854

Figure Lengend Snippet: Fig. 5. p38 MAPK operates downstream of Dishevelleds. (A) F9 cells expressing Rfz1 were treated with siRNAs designed to suppress the expression of Dvl3 for 48 hours and p38 MAPK activity was assayed after 15 minutes of stimulation with Wnt3a. The extent of suppression of Dvl3 is more than 85%. Upper panel represents mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti- Dvl3 (Dvl3) antibodies. (B) F9 cells stably expressing Rfz1 were transfected with Dvl3-GFP2 for 24 hours and p38 MAPK activation was determined. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti-HA (HA) antibodies. (C) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse Dvl3 for 48 hours and the lysates were assayed for cytosolic β-catenin levels after 4 hours of Wnt3a treatment. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), anti-Dvl3 (Dvl3); immunoblots probed with anti-actin antibody (actin) were used as loading controls. (D) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were transfected with either empty vector (–) or with HA- Dvl3-GFP2. After 4 hours of transfection, the transfection medium was replaced with serum medium (15%) containing either DMSO or SB203580 (6 μM) and cultures grown for an additional 20 hours. After 20 hours, the lysates were collected and luciferase assay was performed. Upper panel represents mean values ± s.e.m. from three independent experiments performed in triplicate; lower panel shows representative blots probed with anti-HA (HA) and anti-actin (actin) antibodies. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.

Article Snippet: Rescue experiments were performed by transfecting F9 cells stably expressing Rfz1 and the pTOPFLASH (M50) luciferase reporter with an expression vector harboring siRNA-resistant hp38 (pMT3-p38-HA, Addgene plasmid 12658).

Techniques: Expressing, Activity Assay, Stable Transfection, Transfection, Activation Assay, Luciferase, Western Blot, Plasmid Preparation, Control

Journal: Journal of cell science

Article Title: p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

doi: 10.1242/jcs.032854

Figure Lengend Snippet: Fig. 6. p38 suppresses GSK3β activity. (A) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 μM) for 1 hour prior to the addition of Wnt3a for indicated periods of time. After stimulation, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9-P antibody. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p- GSK3β Ser 9) or with anti-GSK3β (GSK3β) antibodies. (B) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were either transfected with empty vector (–) or with Flag-tagged dominant-negative (DN) mutant of p38 MAPK [p38α (AGF)] for 24 hours. After Wnt3a stimulation for 1 hour, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9- P antibody as described. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p-GSK3β Ser 9), anti-p38 (p38) or with anti-GSK3β (GSK3β) antibodies. (C) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 μM) for 1 hour prior to the addition of Wnt3a for 10 minutes. GSK3β was immunoprecipitated from whole cell lysates and its activity was measured by an in vitro kinase assay. The data represent mean values ± s.e.m. from two independent experiments that are highly reproducible. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.

Article Snippet: Rescue experiments were performed by transfecting F9 cells stably expressing Rfz1 and the pTOPFLASH (M50) luciferase reporter with an expression vector harboring siRNA-resistant hp38 (pMT3-p38-HA, Addgene plasmid 12658).

Techniques: Activity Assay, Stable Transfection, Transfection, Luciferase, Western Blot, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Immunoprecipitation, In Vitro, Kinase Assay, Control

Journal: Journal of cell science

Article Title: p38 mitogen-activated protein kinase regulates canonical Wnt-beta-catenin signaling by inactivation of GSK3beta.

doi: 10.1242/jcs.032854

Figure Lengend Snippet: Fig. 7. Schematic representation of role of p38 MAPK in canonical Wnt–β-catenin signaling pathway. Activation of Fz1 by Wnt3a leads to accumulation of β-catenin, Lef/Tcf- sensitive transcriptional response and primitive endoderm formation in mouse F9 cells through G-proteins and Dishevelleds. p38 MAPK plays a crucial role in canonical signaling by inactivating GSK3β and by operating downstream of Dishevelleds.

Article Snippet: Rescue experiments were performed by transfecting F9 cells stably expressing Rfz1 and the pTOPFLASH (M50) luciferase reporter with an expression vector harboring siRNA-resistant hp38 (pMT3-p38-HA, Addgene plasmid 12658).

Techniques: Activation Assay