Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Ligation of flagellin receptor activates the MAP kinase cascade, which is required for mucin induction. (A) HM3 cells grown in six-well plates were lysed with SDS sample buffer 5 min after the addition of agonist antibody, α-ASGM1. Equal amounts of lysate were run on a 12% SDS gel, transferred to nitrocellulose, immunoblotted with a phospho-specific Erk 1/2 antibody, and visualized by chemiluminescence. Blots were stripped and reprobed with Erk 1/2 antibody to show equal loading. P-Erk Cntrl, positive control from vendor. (B) Effect of MEK 1/2 inhibitor PD98059 (37 μM, 30 min) and dominant negative mutant MEK K97R on MUC 2 promoter activity. (c) Inhibition of Erk phosphorylation by the calcium chelator, BAPTA/AM (30 μM, 45 min). Fold increase expressed as the ratio of phosphorylated Erk to baseline Erk 1/2 as determined by densitometry.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Ligation, SDS-Gel, Positive Control, Dominant Negative Mutation, Activity Assay, Inhibition

Journal:

Article Title: ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor

doi: 10.1073/pnas.161290898

Figure Lengend Snippet: Cartoon depicting events in flagellin-triggered host cell signaling. Flagellin binds to asialoGM1, a membrane glycolipid, which causes the extracellular release of ATP, which then binds to a nucleotide receptor. Downstream events include G-protein activation, the cleavage of PIP2 by PLC, the formation of IP3, Ca2+ mobilization, the phosphorylation of MEK 1/2 and Erk 1/2 [via an unknown calcium binding protein (CBP)], and mucin (MUC 2) transcription. Flagellin-induced signaling bifurcates after Ca2+ mobilization, with ≈50% of the response giving rise to downstream events that are Erk dependent whereas the remaining 50% is Erk independent.

Article Snippet: Phosphospecific anti-Erk 1/2 (mouse monoclonal) was purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Binding Assay

Journal: The journal of pain

Article Title: Serotonin Plays a Key Role in the Development of Opioid-Induced Hyperalgesia in Mice.

doi: 10.1016/j.jpain.2020.12.008

Figure Lengend Snippet: Figure 5. Phosphorylation of ERK2 (p42-MAPK) in the spinal dorsal horn is enhanced following repetitive administering of mor- phine and is reversed by serotonin deprivation via the administering of PCPA. (A−D) Representative immunohistochemistry against pERK and GFAP in the spinal dorsal horns of experimental group mice. (A) Naive group, (B) morphine group, (C) morphine + OND 2.0 mg/kg group and (D) PCPA 150 mg/kg for 8 days + morphine group. White arrows indicate the expression of p-ERK in the spinal dorsal horn. Scale bar: 50 mm in large figures and 20 mm in insets. (E) Western blot analysis. Relative phosphorylation ratios of (F) ERK1 (p44-MAPK) and (G) ERK2 (p42-MAPK). OND (2.0 mg/kg) tended to reduce the phosphorylation ratio of ERK2, although not to a significant level. Neither OND nor serotonin deprivation affected the phosphorylation ratio of ERK1. Saline (control) group: n = 11; morphine group: n = 11; morphine + OND (2.0 mg/kg) group: n = 6; PCPA (150 mg/kg) for 8 days + morphine group: n = 7, respectively.

Article Snippet: The membranes were washed with TBS-T for 5 minutes and incubated overnight at 4°C with the following primary antibodies: anti-human glial fibrillary acidic protein (GFAP; 1:300; Cat. #; M 0761, Dako A/S, Glostrup, Denmark), anti-b-actin (1:5000; Cat. #; A5441, SigmaAldrich, Inc., MO), anti-phospho-p44/42 MAPK (p-ERK 1/ 2) (1:1000, Cat. #;9101, Cell Signaling Technology, Danvers, MA), and anti-p44/42 MAPK (ERK 1/2; 1:1000, Cat. #; 4695, Cell Signaling Technology) diluted in Can Get Signal 1 (Toyobo Co., Ltd., Osaka, Japan).

Techniques: Phospho-proteomics, Immunohistochemistry, Expressing, Western Blot, Saline, Control

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: GluN2B-MDAR scaffolding proteins modulate neutrophil ROS generation. (A) Histograms illustrating ROS generation (quantified by 123-dihydrorhodamine) following stimulation with E. coli and PMA-which is reduced by ZL006 (1–50 μM) in purified primary human neutrophils (n = 7). (B) E. coli -induced ROS in whole blood samples is reduced by ZL006 in a dose-dependent manner (10–50 μM; n = 7). (C) Summary data showing effect of ZL006 on PMA-induced ROS generation in neutrophils, as quantified by 123-dihydrorhodamine. Data expressed as % respective control (mean ± sem; n = 7 subjects; *p = 0.01, ANOVA). (D) shRNA knockdown of GluN1B is associated with reduced nNOS protein expression in differentiated HL60 neutrophil-like cells. (E) PMA-induced ROS production (quantified by 123-dihydrorhodamine) is attenuated in bone-marrow derived neutrophils from wild-type and homozygous PSD-95 deficient. (F) Population data for ROS in wild-type and homozygous PSD-95 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.02 unpaired t -test). (G) ERK phosphorylation in primary human neutrophils is reduced by GluN2B antagonist Co 101244 following PMA-stimulation phospho-flow cytometry in CD16+ cells, human whole blood (mean ± sem; n = 5 experiments; *p < 0.05; two-way (drug × time) ANOVA with Tukey posthoc comparison). Immunoblot above graph shows similar results obtained in five separate experiments. Numbers below123-dihydrorhodamine immunoblot denote minutes after PMA stimulation (in the presence/absence of GluN2B subunit antagonist Co 101244) or phosphate-buffered saline control. (H) PMA-induced ROS production is attenuated in bone-marrow derived neutrophils from wild-type and homozygous SAP102 deficient mice. (I) Population data for wild-type and homozygous SAP-102 deficient bone marrow derived neutrophils (mean ± sem; n = 6–7 mice/group; *p = 0.04 unpaired t -test).

Article Snippet: Membranes were incubated with the following primary antibodies (from Santa Cruz Biotechnology, Heidelberg, Germany, unless otherwise stated): GluN1 (sc-9056), NE-dlg (SAP102; sc-134,400), PSD-95 (sc-28,941), ERK (1:1000; Cell Signaling Technology, Leiden; #4695), phospho-ERK (1:1000; Cell Signaling Technology, Leiden; #4370), AKT (1:1000; Cell Signaling Technology, Leiden; #9272), phospho-AKT (S473) (1:1000; Cell Signaling Technology, Leiden; #9271), NOX-2 (1:1000; Abcam, Oxford; ab129068).

Techniques: Scaffolding, Purification, Control, shRNA, Knockdown, Expressing, Derivative Assay, Phospho-proteomics, Flow Cytometry, Comparison, Western Blot, Saline

Journal: EBioMedicine

Article Title: NMDA receptor modulation of glutamate release in activated neutrophils

doi: 10.1016/j.ebiom.2019.08.004

Figure Lengend Snippet: NMDAR modulation of neutrophil survival. (A) Annexin V versus propidium density plots to quantify constitutive apoptosis in HL60-neutrophil like cell line, comparing control (scrambled shRNA) with shRNA knockdown of GluN1 (representative plots of n = 3 experiments). Application of MK801, a non-competitive NMDAR blocker, failed to reduce ROS in cells where GluN1 expression was reduced. In contrast, MK801 increased baseline ROS in scrambled cells. (B) Immunoblot for NADPH oxidase (isoform-2; 1:1000; Cell Signalling) in differentiated, scrambled or GluN1deficient HL60-neutrophil like cell line. GAPDH served as loading control. Densitometry ratios summarised in graph; n = 3; * p = 0.02, by Students t -test. (C) Immunoblot for AKT and phosphorylation of AKT (1:1000;Cell Signalling) in differentiated, scrambled or GluN1deficient HL60-neutrophil like cell line. GAPDH served as loading control (Densitometry ratios summarised in graph; n = 3; * p = 0.04, by Students t -Test). (D) Immunoblot for ERK and phosphorylation of ERK (1:1000; Cell Signalling) in differentiated, scrambled or GluN1deficient HL60-neutrophil like cell line. Total GAPDH (cytoplasmic and nuclear) served as loading control, hence dual band. (Densitometry ratios are summarised in graph; n = 3).

Article Snippet: Membranes were incubated with the following primary antibodies (from Santa Cruz Biotechnology, Heidelberg, Germany, unless otherwise stated): GluN1 (sc-9056), NE-dlg (SAP102; sc-134,400), PSD-95 (sc-28,941), ERK (1:1000; Cell Signaling Technology, Leiden; #4695), phospho-ERK (1:1000; Cell Signaling Technology, Leiden; #4370), AKT (1:1000; Cell Signaling Technology, Leiden; #9272), phospho-AKT (S473) (1:1000; Cell Signaling Technology, Leiden; #9271), NOX-2 (1:1000; Abcam, Oxford; ab129068).

Techniques: Control, shRNA, Knockdown, Expressing, Western Blot, Phospho-proteomics