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: 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

Journal: bioRxiv

Article Title: Deep Invaginations of Nuclear Envelope Coordinate Spatial Organization of Chromatin in Epithelium

doi: 10.64898/2026.03.10.710762

Figure Lengend Snippet: (A) The volcano plot of the differentially expressed genes (DEGs) in mature 7 d grown epithelium in comparison to developing 2 d grown epithelium. MAPK-signaling -associated genes highlighted in red. (B) Ingenuity pathway analysis (IPA) pathway generator -derived map presenting affected nuclear mechanotransduction and NE -associated components and their downstream actors. Red and green indicate up- and down-regulation, respectively. Purple lines indicate statistically significantly affected components (Abs(log2FC)) >1 and pAdj <0.05). (C) ClusterProfiler KEGG pathway over-representation “MAPK signaling pathway” analysis of the 7 d -grown mature epithelium showing significantly affected biological processes in the 7-d grown mature epithelium in comparison to the 2 d-grown epithelium. (D) IPA pathway generator -derived map showing affected components of MAPK-signaling. Red indicates up-regulated, and green indicates down-regulated. Purple lines indicate statistically significantly affected components (Abs(log2FC)) >1 and pAdj <0.05). (E) STRING analysis of MAPK-associated DEGs in the 7 d grown epithelium, indicating functional enrichment in three clusters within interconnected networks. (F) Transcription factor (TF) motif analysis of transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) of 2 d and 7 d grown cells indicates global decreased accessibility of TF motifs, highlighting significant suppression of MAPK/ERK-associated motifs in the mature epithelium. Box-and-whiskers plots presenting (G) intracellular normalized fluorescence intensity distribution, and (H) nucleo-cytoplasmic ratio of activated phosphorylated ERK1/2 (phospho-ERK, p44/42 MAPK) at 2, 4, and 7 d post-seeding. One-way ANOVA with Dunnett’s multiple comparisons test indicates statistical significance, n=three independent biological replicates. Box-and-whisker plots represent the 25 th –75 th percentiles as boxes, the median as a line within the box, and whiskers indicating the minimum and maximum values.

Article Snippet: Active MAPK was detected by using a mouse monoclonal Ab identifying the phosphorylated p44/42 MAPK (1:400, ERK1/2, Thr202/Tyr204 [E10], #9106, Cell Signaling Technology, MA, USA).

Techniques: Comparison, Derivative Assay, Functional Assay, Next-Generation Sequencing, Fluorescence, Whisker Assay