Journal: International Journal of Alzheimer's Disease

Article Title: Evidence for Elevated Cerebrospinal Fluid ERK1/2 Levels in Alzheimer Dementia

doi: 10.4061/2011/739847

Figure Lengend Snippet: Detection of ERK1, ERK2, and doubly phosphorylated ERK2 in a pooled human CSF sample. Following prefractionation of 3.25 mL of pooled CSF on an Off-gel fractionator, the proteins with isoelectric points in the pH range from ~6.5 to ~8.5 were pooled, precipitated with trichloroacetic acid, and analyzed by SDS-PAGE/Western blot with the indicated antibodies. A sample corresponding to ~0.54 mL of the original CSF-pool was loaded on each lane. (1) Magic mark protein standard, (2) CSF pool, (3) positive control (cell lysate of okadaic-acid-treated SH-SY5Y cells).

Article Snippet: To obtain a suitable reference sample for calculating the standard curves, recombinant activated His-tagged ERK2 (Calbiochem) was initially spiked into a pooled human CSF sample at a concentration of 480 ng/mL (“CSF-stabilized ERK2 stock solution”).

Techniques: SDS Page, Western Blot, Positive Control

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK2-mediated phosphorylation of mGluR1a. a GST-fusion proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue (CBB) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min (b–e) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images (b–d) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Autoradiography, Staining, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Migration

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: Phosphorylation of mGluR1a-CT2 by ERK2. a Amino acid sequence analysis of mGluR1a-CT2(P1001-L1199). Eight ERK2 phosphorylation motifs (T/SP) are highlighted in red. Potential phosphorylation sites include T1064 (1), S1098 (2), T1143 (3), S1147 (4), T1151 (5), S1154 (6), S1169 (7), and T1178 (8). b A representative phosphoamino acid analysis showing GST-mGluR1a-CT2 phosphorylation at serine (pSer), but not threonine (pThr) and tyrosine (pTyr), residues. c, d Phosphorylation of mGluR1a-CT2 at pS/TP (c) and pTP (d) motifs by active ERK2. e Five mutants (M1–5) derived from mGluR1a-CT2(P1001-L1199). Site-directed mutants include mutations of T1064/S1098 to alanines (M1), T1064/S1098/T1143/S1147 to alanines (M2), T1064/S1098/T1143/S1147/T1151/S1154 to alanines (M3), four serines (S1098/S1147/S1154/S1169) to alanines (M4), and four threonines (T1064/T1143/T1151/T1178) to alanines (M5). e Phosphorylation of mGluR1a-CT2 mutants and WT at pS/TP by active ERK2. Note that no signal was detected in the M4 mutant. g Phosphorylation of GST-mGluR1a-CT2 and Elk-1 at PXpSP by active ERK2. Phosphorylation of CT2 and Elk-1 was detected by immunoblot (IB) with a phosphomotif antibody against pS/TP (c, f), pTP (d), or PXpSP (g)

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Sequencing, Phosphoamino Acid Analysis, Derivative Assay, Mutagenesis, Western Blot

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK2 binding to mGluR1a. a In vitro binding assays with immobilized GST-fusion proteins containing distinct intracellular domains of mGluR1a and purified ERK2. Inactive ERK2 proteins were used in these assays. The inactive state of ERK2 was confirmed by the lack of phosphorylation signals from ERK2 proteins when tested in immunoblots (IB) with a phospho-specific antibody against pERK1/2. b In vitro binding assays with immobilized mGluR1a CT1 fragments and inactive or active ERK2. c GST-fusion proteins containing different fragments of mGluR1a CT1. d, e In vitro binding assays with immobilized GST-fusion mGluR1a-CT1a–c proteins and inactive (d) or active (e) ERK2. Note that CT1a but not CT1b and CT1c precipitated ERK2 and pERK2. ERK2 and pERK2 proteins bound to GST-fusion proteins were visualized with immunoblots using the specific antibodies against ERK2 and pERK1/2, respectively

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Binding Assay, In Vitro, Purification, Western Blot

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: Interactions of ERK2 with mGluR1a in rat cerebellar neurons. a, b Immunoblot detection of mGluR1a (a) and mGluR5 (b) proteins in synaptic membranes extracted from the cerebellum and striatum. Note that a smaller amount of cerebellar proteins (2.5 µg) than that of striatal proteins (25 µg) was loaded (a). Major dimer bands around 250 kDa and weak monomer bands (130–140 kDa) were seen for mGluR1a in the cerebellum and striatum, while mGluR5 bands (mainly dimers) were seen in the striatum but not cerebellum. c, d Immunoblot detection of ERK1/2 (c) and pERK1/2 (d) proteins in whole-cell homogenates and synaptic plasma membranes of cerebellar neurons (12.5 µg per lane). Note that ERK2 and pERK2 were expressed at a higher level than respective ERK1 and pERK1 in synaptic locations. e Coimmunoprecipitation (IP) assays of ERK1/2 and mGluR1a with the anti-mGluR1a antibody (Ab). Note that ERK2 clearly existed in mGluR1a precipitates (lane 5). Lanes 3 and 4 showed no specific bands due to the lack of a precipitating antibody (L3) and the use of an irrelevant IgG (L4). f Reverse coimmunoprecipitation assays of ERK1/2 and mGluR1a with the anti-ERK1/2 antibody. g Coimmunoprecipitation of pERK1/2 and mGluR1a. Synaptic proteins solubilized from enriched synaptic plasma membranes of the rat cerebellum were used in above IP assays. Precipitated proteins were visualized by immunoblots (IB) with indicated antibodies

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Western Blot

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK-mediated phosphorylation of mGluR1a in rat cerebellar neurons. a–c Phosphorylation of mGluR1a at ERK-preferred motifs, including S/TP (a), TP (b), and PXSP (c). Cerebellar mGluR1a was immunopurified by the anti-mGluR1a antibody. Phosphorylation signals at three phosphomotifs (pS/TP, pTP, and PXpSP) in immunopurified mGluR1a were detected by immunoblots (IB) with indicated antibodies. d Dephosphorylation of mGluR1a PXSP phosphorylation by λ-protein phosphatase (λ-PP). Cerebellar slices were incubated with λ-PP (200–400 units) for 1 h at 30 °C. Note that PXSP phosphorylation was dephosphorylated by λ-PP. e Representative immunoblots illustrating effects of U0126 on phosphorylation of mGluR1a at PXSP and TP motifs and on pERK1/2-mGluR1a and ERK1/2-mGluR1a interactions in cerebellar neurons. f, g Quantifications of effects of U0126 on mGluR1a PXSP (f) and TP (g) phosphorylation. h Quantifications of effects of U0126 on pERK2-mGluR1a and ERK2-mGluR1a interactions. Cerebellar slices were incubated with U0126 (0.5 or 5 µM) for 30 min at 30 °C (e–h). Slices were collected for mGluR1a immunoprecipitation (IP). Phosphorylation levels of mGluR1a at PXSP and TP and proteins bound to immunopurified mGluR1a (pERK1/2 and ERK1/2) were visualized by immunoblots. Values in graphs were measured as ratios of PXpSP to mGluR1a (f), pTP to mGluR1a (g), pERK2 to mGluR1a (h), and ERK2 to mGluR1a (h). All values were analyzed by one-way ANOVA: PXpSP (f), F(2, 7) = 5.05, p < 0.05; pTP (g), F(2, 7) = 0.26, p > 0.05; pERK2 (h), F(2, 7) = 37.44, p < 0.05; and ERK2 (h), F(2, 7) = 14.67, p < 0.05. Data are presented as means ± SEM (n = 3–4 per group). *p < 0.05 versus vehicle

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Western Blot, De-Phosphorylation Assay, Incubation, Immunoprecipitation

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK regulates the mGluR1-mediated IP3 production in rat cerebellar neurons. a A rapid elevation of cytosolic IP3 levels following application of DHPG (100 µM). b Effects of the mGluR1 antagonist 3-MATIDA on the DHPG-stimulated IP3 formation. Note that 3-MATIDA completely blocked IP3 responses to DHPG. c Effects of U0126 on the DHPG-stimulated IP3 production. Note that U0126 at 5 µM significantly reduced IP3 responses to DHPG. d Tat-fusion mGluR1a interaction-dead (Tat-mGluR1a-id) and sequence-scrambled control (Tat-mGluR1a-con) peptides. The underlined letters (LNIFRRKK) represent core hydrophobic and basic residues in a D domain-like sequence of mGluR1a-CT. e, f Effects of Tat-fusion peptides on the ERK2-mGluR1a association (e) and DHPG-stimulated IP3 production (f). Note that Tat-mGluR1a-id but not Tat-mGluR1a-con peptides reduced the ERK2-mGluR1a association and IP3 responses to DHPG. Experiments were carried out in rat cerebellar slices. 3-MATIDA (10 µM) or U0126 (0.5 or 5 µM) was applied 30 min before and during 20–25 s incubation of DHPG (100 µM) (b, c). Tat-fusion peptides (5 µM in e and 2 or 5 µM in f) were added to cerebellar slices for 30 min. Slices were then used for coimmunoprecipitation detection of the ERK2-mGluR1a association (e) or IP3 measurements (f). Values in the panel a were analyzed by Student’s t test (t = 3.21, p < 0.05). Values in panels b, c, e, and f were analyzed by one-way ANOVA: 3-MATIDA + DHPG (b), F(3, 16) = 35.05, p < 0.05; U0126 + DHPG (c), F(5, 26) = 19.10, p < 0.05; ERK2-mGluR1a interactions (e), F(2, 15) = 7.50, p < 0.05; and Tat-peptides + DHPG (f), F(9, 29) = 9.55, p < 0.05. Data are presented as means ± SEM (n = 4–7 per group). *p < 0.05 versus vehicle (a, e) or vehicle + vehicle (b, c, and f). +p < 0.05 versus vehicle + DHPG (b, c, and f)

Article Snippet: Briefly, His-tagged active ERK2-FL with pT185/pY187 (Millipore, Billerica, MA; 20 ng) or His-tagged inactive ERK2-FL (Millipore; 20 ng) was equilibrated to binding buffer containing 200 mM NaCl, 0.2 % Triton X-100, 0.1 mg/ml bovine serum albumin (BSA), and 50 mM Tris, pH, 7.5.

Techniques: Sequencing, Incubation

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK2-mediated phosphorylation of mGluR1a. a GST-fusion proteins containing distinct intracellular domains of rat mGluR1a. b A representative autoradiograph illustrating the ERK2-induced phosphorylation of GST-mGluR1a-CT2 and Elk-1 but not GST and other mGluR1a fragments. A gel staining with Coomassie Brilliant Blue (CBB) was present below to show protein loading. Note that the CT2 segment was markedly phosphorylated by ERK2. c A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 by active but not inactive ERK2. d A representative autoradiograph above a CBB staining illustrating phosphorylation of GST-mGluR1a-CT2 in the presence but not absence of ATP. e CIP-mediated dephosphorylation of ERK2-phosphorylated GST-mGluR1a-CT2. All phosphorylation reactions were carried out at 30 °C for 30 min (b–e) followed by dephosphorylation reactions (e). The reactions were then subjected to gel electrophoresis followed by autoradiography. Solid and open arrows indicate phosphorylated GST-mGluR1a-CT2 (pGST-mGluR1a-CT2) and Elk-1, respectively. Red stars in CBB staining images (b–d) indicate the CT2 and Elk-1 bands that were phosphorylated by ERK2. Note that these bands smeared and shifted upward as a result of slower migration due to phosphorylation

Article Snippet: GST or GST-fusion proteins were incubated with GST-tagged active ERK2 (Millipore, 25 ng) or GST- or His-tagged inactive ERK2 (Millipore, 25 ng) for 30 min at 30 °C in a reaction buffer (25 μl) containing 10 mM HEPES pH 7.4, 10 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 50 μM ATP, and 2.5 μCi/tube [γ- 32 P]ATP (~3000 Ci/mmol, PerkinElmer, Waltham, MA) with an Elk-1 fusion protein (Cell Signaling, Danvers, MA).

Techniques: Phospho-proteomics, Autoradiography, Staining, De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Migration

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: Phosphorylation of mGluR1a-CT2 by ERK2. a Amino acid sequence analysis of mGluR1a-CT2(P1001-L1199). Eight ERK2 phosphorylation motifs (T/SP) are highlighted in red. Potential phosphorylation sites include T1064 (1), S1098 (2), T1143 (3), S1147 (4), T1151 (5), S1154 (6), S1169 (7), and T1178 (8). b A representative phosphoamino acid analysis showing GST-mGluR1a-CT2 phosphorylation at serine (pSer), but not threonine (pThr) and tyrosine (pTyr), residues. c, d Phosphorylation of mGluR1a-CT2 at pS/TP (c) and pTP (d) motifs by active ERK2. e Five mutants (M1–5) derived from mGluR1a-CT2(P1001-L1199). Site-directed mutants include mutations of T1064/S1098 to alanines (M1), T1064/S1098/T1143/S1147 to alanines (M2), T1064/S1098/T1143/S1147/T1151/S1154 to alanines (M3), four serines (S1098/S1147/S1154/S1169) to alanines (M4), and four threonines (T1064/T1143/T1151/T1178) to alanines (M5). e Phosphorylation of mGluR1a-CT2 mutants and WT at pS/TP by active ERK2. Note that no signal was detected in the M4 mutant. g Phosphorylation of GST-mGluR1a-CT2 and Elk-1 at PXpSP by active ERK2. Phosphorylation of CT2 and Elk-1 was detected by immunoblot (IB) with a phosphomotif antibody against pS/TP (c, f), pTP (d), or PXpSP (g)

Article Snippet: GST or GST-fusion proteins were incubated with GST-tagged active ERK2 (Millipore, 25 ng) or GST- or His-tagged inactive ERK2 (Millipore, 25 ng) for 30 min at 30 °C in a reaction buffer (25 μl) containing 10 mM HEPES pH 7.4, 10 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 50 μM ATP, and 2.5 μCi/tube [γ- 32 P]ATP (~3000 Ci/mmol, PerkinElmer, Waltham, MA) with an Elk-1 fusion protein (Cell Signaling, Danvers, MA).

Techniques: Phospho-proteomics, Sequencing, Phosphoamino Acid Analysis, Derivative Assay, Mutagenesis, Western Blot

Journal: Molecular neurobiology

Article Title: Synaptic ERK2 Phosphorylates and Regulates Metabotropic Glutamate Receptor 1 In Vitro and in Neurons

doi: 10.1007/s12035-016-0225-4

Figure Lengend Snippet: ERK2 binding to mGluR1a. a In vitro binding assays with immobilized GST-fusion proteins containing distinct intracellular domains of mGluR1a and purified ERK2. Inactive ERK2 proteins were used in these assays. The inactive state of ERK2 was confirmed by the lack of phosphorylation signals from ERK2 proteins when tested in immunoblots (IB) with a phospho-specific antibody against pERK1/2. b In vitro binding assays with immobilized mGluR1a CT1 fragments and inactive or active ERK2. c GST-fusion proteins containing different fragments of mGluR1a CT1. d, e In vitro binding assays with immobilized GST-fusion mGluR1a-CT1a–c proteins and inactive (d) or active (e) ERK2. Note that CT1a but not CT1b and CT1c precipitated ERK2 and pERK2. ERK2 and pERK2 proteins bound to GST-fusion proteins were visualized with immunoblots using the specific antibodies against ERK2 and pERK1/2, respectively

Article Snippet: GST or GST-fusion proteins were incubated with GST-tagged active ERK2 (Millipore, 25 ng) or GST- or His-tagged inactive ERK2 (Millipore, 25 ng) for 30 min at 30 °C in a reaction buffer (25 μl) containing 10 mM HEPES pH 7.4, 10 mM MgCl 2 , 1 mM Na 3 VO 4 , 1 mM dithiothreitol (DTT), 0.1 mg/ml BSA, 50 μM ATP, and 2.5 μCi/tube [γ- 32 P]ATP (~3000 Ci/mmol, PerkinElmer, Waltham, MA) with an Elk-1 fusion protein (Cell Signaling, Danvers, MA).

Techniques: Binding Assay, In Vitro, Purification, Phospho-proteomics, Western Blot

Journal: Journal of fluorescence

Article Title: Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum.

doi: 10.1007/s10895-016-1840-1

Figure Lengend Snippet: Fig. 5 Spectrofluorometric comparison of the ERK aptamer titration in TE buffer with OG and PG without (panels a and c) and with 500 ng/ml of recombinant human ERK2 protein (panels b and d) added in 100 μl of TE buffer to each cuvette following the readings taken in panels a and c and mixing for 15 min at RT. The increase in fluorescence indicated by the asterisk was not reproducible in subsequent experiments (data not shown), but is reported because it may have been transient. All emission scans were from 500 or 505 nm to 600 nm

Article Snippet: Recombinant human ERK2 protein was purchased from Novus Biologicals Inc. (Littleton, CO) and kept frozen at -20 °C until thawed at room temperature (RT ~ 25 °C) just prior to use in experiments.

Techniques: Comparison, Titration, Recombinant, Fluorescence