Recombinant:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) .. T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 .
Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
Marker:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) .. T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 .
Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
MTT Assay:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) .. T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 .
Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
Control:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) .. T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 .
Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
Injection:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
Animal Model:Article Title: Tissue-type Plasminogen Activator Protects Neurons from Excitotoxin-induced Cell Death via Activation of the ERK 1/2 -CREB-ATF3 Signaling Pathway
Article Snippet: Experiments were approved by the Institutional Animal Care & Use Committee of Emory University, Atlanta GA. Recombinant murine tPA and inactive tPA (itPA) with an alanine for serine substitution at the active site Ser481 (S481A) were purchased from Molecular Innovations (Novi, MI). .. Other reagents were human recombinant tPA (rtPA; Genentech; San Francisco, CA), the Receptor-Associated Protein (RAP; kindly provided by Dr. Dudley K. Strickland, University of Maryland), the NMDAR antagonist MK-801, the NR2A antagonist TCN-201, the MEK 1⁄2 inhibitor SL327, the NR2B antagonist Ro 25 6981 and bicuculline (Tocris Bioscience; Ellisville, MO), NMDA (Sigma-Aldrich; St. Louis, MO), antibodies against NR2A phosphorylated at Y1325, total NR2A and Atf3 (Abcam; Cambridge, MA), NR2B phosphorylated at Tyr1472 and total NR2B (Sigma-Aldrich; St. Louis, MO), CREB phosphorylated at Ser 133, total CREB, ERK 1⁄2 phosphorylated at Thr202/Tyr204, total ERK 1⁄2 and cleaved caspase-3 (Cell Signaling Technology Inc; Danvers, MA), and anti-actin (Santa Cruz Biotechnology; Santa Cruz, CA); the nuclear marker 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes; Grand Island, NY), and the 3-(4,5 Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Manassas, VA), and lactate dehydrogenase (LDH) release assays (Roche, Indianapolis, IN), the TrkB inhibitor K-252a (Calbiochem, Rockland, Massachusetts), and Atf3 siRNA (E-058604-00-0010) and a non-targeting control (D-001910-10-20;Thermo Scientific Dharmacon; Lafayette, CO), and primers for Gapdh: Mm99999915_g1 and Atf3: Mm00476032_m1 (Applied Biosystems; Carlsbad, CA) Intracerebral injection of NMDA and animal model of cerebral ischemia T4 mice and their corresponding Wt littermate controls (n = 12 per experimental group) were anesthetized with 4% chloral hydrate (400 mg/kg/IP) and injected with 1 ml of NMDA 50 mM, using a microsyringe pump (World Precision Instruments; Sarasota, Fl), at an infusion rate of 50 nL/min (total infusion time: 20 minutes), at the following coordinates: bregma: 0.26 mm, mediolateral: 2 mm, dorsoventral: 3 mm 16 . ..
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