Incubation:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
Article Title: Sleep Deprivation-Induced Anxiety Alleviated by Oral Administration of 4-Aminopyridine in Male Mice.
Article Snippet: Proteins were then separated by SDS-PAGE on a 10% gel and then transferred onto PVDF membranes (Millipore). .. The membranes were blocked with 3% BSA in PBS for 1 h before incubation overnight at 4◦Cwith the following antibodies: anti-AMPA-R (Cell Signaling-2460, diluted 1:1000), anti-NMDAR (ab-14596, Abcam:500F, diluted), (sc-130349, diluted 1:500), and anti-β-actin (sc-47778, diluted 1:500). .. For detection, secondary antibodies specific to the primary antibodieswere used, including anti-AMPA-R (Sell Signaling, mouse monoclonal antibody), antiNMDA-R (Abcam, rabbit antibody), anti-TNF-α (SANTA CRUZ, mouse monoclonal antibody), and anti-β-actin monoclonal antibody (SANTAe). β-actin protein served as a housekeeping protein.
Article Title: NADPH oxidase-1 suppression prolongs the antidepressant-like effect of ketamine.
Article Snippet: Protein samples (5-10 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–15% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). .. Membranes were blocked with 1% blocking buffer (Perfect-block, MoBiTec) in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies: anti-NOX-1 (1:1000, ab131088, Abcam (for primary neuron samples); 1:1000, GTX103888, GeneTex (for crude brain samples)), anti-pan-AMPAR (1:1000, 2460, Cell Signaling Technology), antiGAD1 (1:1000, 41318, Cell Signaling Technology), anti-Phospho-mTOR (1:1000, 2971, Cell Signaling Technology), anti-Phospho-ERK1/2 (1:1000, 9101, Cell Signaling Technology), anti-NMDA receptor (GluN1, 1:1000, D65B7, Cell Signaling Technology), anti-GAPDH (1:2000, 14C10, Cell Signaling Technology) and anti-β-actin (1:10000, A5441, Sigma). .. Membranes were washed in TBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (Peroxidase-conjugated AffiniPure Goat anti-rabbit or anti-mouse, 1:1000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature.
Article Title: Sleep Deprivation‐Induced Anxiety Alleviated by Oral Administration of 4‐Aminopyridine in Male Mice
Article Snippet: Proteins were then separated by SDS‐PAGE on a 10% gel and then transferred onto PVDF membranes (Millipore). .. The membranes were blocked with 3% BSA in PBS for 1 h before incubation overnight at 4°C with the following antibodies: anti‐AMPA‐R (Cell Signaling‐2460, diluted 1:1000), anti‐NMDA‐R (ab‐14596, Abcam:500F, diluted), (sc‐130349, diluted 1:500), and anti‐β‐actin (sc‐47778, diluted 1:500). .. For detection, secondary antibodies specific to the primary antibodies were used, including anti‐AMPA‐R (Sell Signaling, mouse monoclonal antibody), anti‐NMDA‐R (Abcam, rabbit antibody), anti‐TNF‐α (SANTA CRUZ, mouse monoclonal antibody), and anti‐β‐actin monoclonal antibody (SANTAe). β‐actin protein served as a housekeeping protein.
Article Title: Integrin α 3 is required for high‐frequency repetitive transcranial magnetic stimulation‐induced glutamatergic synaptic transmission in mice with ischemia
Article Snippet: .. Membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: integrin α 3 (1:1000, 66,070–1, Proteintech), GluN2B (1:1000, 21,920‐1‐AP, Proteintech), β ‐actin (1:1000, SD0034, SIMUWU), GluN2A (1:1000, 19,953‐1‐AP, Proteintech), GluA2/3/4 (1:1000, 2460, Cell Signaling), GluA1 (1:1000, 67,642‐1‐Ig, Proteintech), GAD65 (1:1000, 20,746‐1‐AP, Proteintech), and vGlut1 (1:1000, ab227805, Abcam). ..
Expressing:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
Control:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
Concentration Assay:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
Activation Assay:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
Phospho-proteomics:Article Title: Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging
Article Snippet: Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. .. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA A , Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). .. The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature.
other:
Article Title: Inflammation-responsive framework nucleic acid-enabled effective intestinal accumulation for ulcerative colitis therapy.
Article Snippet: Therapeutic oligonucleotides targeting GATA3 have emerged as promising immune-modulating drugs for treating ulcerative colitis (UC).. However, achieving effective in vivo delivery of therapeutic oligonucleotides to selectively modulate target cells is challenging due to the intestinal barrier and cellular heterogeneity.. Here, we demonstrated the intravenous delivery of GATA3-targeted oligonucleotide (hgd40 DNAzyme) to the intestine utilizing tetrahedral framework nucleic acids (tFNAs).
Blocking Assay:Article Title: NADPH oxidase-1 suppression prolongs the antidepressant-like effect of ketamine.
Article Snippet: Protein samples (5-10 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–15% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). .. Membranes were blocked with 1% blocking buffer (Perfect-block, MoBiTec) in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies: anti-NOX-1 (1:1000, ab131088, Abcam (for primary neuron samples); 1:1000, GTX103888, GeneTex (for crude brain samples)), anti-pan-AMPAR (1:1000, 2460, Cell Signaling Technology), antiGAD1 (1:1000, 41318, Cell Signaling Technology), anti-Phospho-mTOR (1:1000, 2971, Cell Signaling Technology), anti-Phospho-ERK1/2 (1:1000, 9101, Cell Signaling Technology), anti-NMDA receptor (GluN1, 1:1000, D65B7, Cell Signaling Technology), anti-GAPDH (1:2000, 14C10, Cell Signaling Technology) and anti-β-actin (1:10000, A5441, Sigma). .. Membranes were washed in TBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (Peroxidase-conjugated AffiniPure Goat anti-rabbit or anti-mouse, 1:1000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature.
Saline:Article Title: NADPH oxidase-1 suppression prolongs the antidepressant-like effect of ketamine.
Article Snippet: Protein samples (5-10 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 4–15% gradient gels (Mini-PROTEAN TGX; Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore). .. Membranes were blocked with 1% blocking buffer (Perfect-block, MoBiTec) in tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies: anti-NOX-1 (1:1000, ab131088, Abcam (for primary neuron samples); 1:1000, GTX103888, GeneTex (for crude brain samples)), anti-pan-AMPAR (1:1000, 2460, Cell Signaling Technology), antiGAD1 (1:1000, 41318, Cell Signaling Technology), anti-Phospho-mTOR (1:1000, 2971, Cell Signaling Technology), anti-Phospho-ERK1/2 (1:1000, 9101, Cell Signaling Technology), anti-NMDA receptor (GluN1, 1:1000, D65B7, Cell Signaling Technology), anti-GAPDH (1:2000, 14C10, Cell Signaling Technology) and anti-β-actin (1:10000, A5441, Sigma). .. Membranes were washed in TBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (Peroxidase-conjugated AffiniPure Goat anti-rabbit or anti-mouse, 1:1000, Jackson ImmunoResearch Laboratories) for 1 h at room temperature.
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