Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells

doi: 10.3390/ijms262110311

Figure Lengend Snippet: Surface ChAT staining and flow cytometric analyses in a neuroblastoma cell line compared to three lung cancer cell lines. The left panel shows dot plots of Live/Dead dye staining versus ChAT staining in SH-SY5Y neuroblastoma cells. The upper dot plot shows unstained cells, the middle plot shows results after blocking the antibody with recombinant human ChAT protein, and the lower plot indicates the percentage of SH-SY5Y cells positive for ChAT staining using the ChAT antibody alone. The values in the dot plots represent mean ± SD of triplicates. The right panel presents histograms for the small cell lung cancer lines H82 and H69, and the lung adenocarcinoma cell line A549, stained similarly to the left panel. Notably, plasma membrane integrity was also assessed in the lung cancer cell lines using Live/Dead dye staining. All cells exhibited intact plasma membranes, as no major signal was detected in quadrants Q1 and Q2. Abbreviations: ChAT = choline acetyltransferase; APC anti-ChAT Ab = allophycocyanin-conjugated anti-ChAT antibody.

Article Snippet: Three different unconjugated anti-ChAT primary antibodies were used for confocal imaging: a monoclonal mouse anti-ChAT antibody (MAB3447, R&D Systems, Bio-Techne, Stockholm, Sweden), which targets a region between Ala2–Pro630 (Accession # NP_066266 ); a rabbit polyclonal anti-ChAT antibody ( PAB14536 , Abnova, Taipei, Taiwan); and another rabbit polyclonal anti-ChAT antibody (AB143, Millipore via Merck).

Techniques: Staining, Blocking Assay, Recombinant, Clinical Proteomics, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells

doi: 10.3390/ijms262110311

Figure Lengend Snippet: Whole-cell ChAT staining and flow cytometric analyses in a neuroblastoma cell line compared to three lung cancer cell lines. The left panel shows dot plots of side scatter area versus ChAT staining in SH-SY5Y neuroblastoma cells following membrane permeabilization, allowing the anti-ChAT antibody to access both surface and intracellular ChAT protein, thereby reflecting whole-cell ChAT staining. The upper dot plot shows unstained cells, the middle plot shows cells incubated with a pre-blocked anti-ChAT antibody, and the lower plot indicates the percentage of SH-SY5Y cells positive for ChAT expression. The right panel presents histograms for the small cell lung carcinoma lines H82 and H69, and the lung adenocarcinoma cell line A549, analyzed similarly to the left panel. Notably, ChAT staining in the lung carcinoma cell lines reflects only intracellular localization, as these cells lacked surface-localized ChAT (as shown in ). A comparison of ChAT staining in SH-SY5Y cells (79 ± 3%) with the extracellular ChAT data in (66 ± 1%) indicates that, in contrast to the lung carcinoma cell lines, most of the ChAT protein in SH-SY5Y cells is localized extracellularly. The values in the dot plots represent mean ± SD of triplicates. ChAT = choline acetyltransferase; APC anti-ChAT Ab = allophycocyanin-conjugated anti-ChAT antibody.

Article Snippet: Three different unconjugated anti-ChAT primary antibodies were used for confocal imaging: a monoclonal mouse anti-ChAT antibody (MAB3447, R&D Systems, Bio-Techne, Stockholm, Sweden), which targets a region between Ala2–Pro630 (Accession # NP_066266 ); a rabbit polyclonal anti-ChAT antibody ( PAB14536 , Abnova, Taipei, Taiwan); and another rabbit polyclonal anti-ChAT antibody (AB143, Millipore via Merck).

Techniques: Staining, Membrane, Incubation, Expressing, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Comparative Analysis of Cholinergic Machinery in Carcinomas: Discovery of Membrane-Tethered ChAT as Evidence for Surface-Based ACh Synthesis in Neuroblastoma Cells

doi: 10.3390/ijms262110311

Figure Lengend Snippet: Relative surface and total protein expression of cholinergic markers in SH-SY5Y neuroblastoma cells. Protein levels were quantified based on fluorescence intensity (FI) obtained from surface and whole-cell flow cytometry data ( and ). Panel ( A ) presents relative fluorescence intensity (RFI) values normalized to controls: for ChAT, normalization was performed against cells incubated with a blocked anti-ChAT antibody, while for AChE, BChE, α7-nAChR, and M1 mAChR, normalization was done using cells stained only with the Live/Dead dye. Panel ( B ) depicts the fold difference between total cellular and surface-localized protein expression, calculated by dividing FI from whole-cell staining by FI from surface staining. Bar graphs represent the mean ± standard deviation (SD). Abbreviations: ChAT = choline acetyltransferase; AChE = acetylcholinesterase; BChE = butyrylcholinesterase; α7 AChR = α7-subtype of nicotinic acetylcholine receptor; M1 AChR = M1-subtype of muscarinic acetylcholine receptor.

Article Snippet: Three different unconjugated anti-ChAT primary antibodies were used for confocal imaging: a monoclonal mouse anti-ChAT antibody (MAB3447, R&D Systems, Bio-Techne, Stockholm, Sweden), which targets a region between Ala2–Pro630 (Accession # NP_066266 ); a rabbit polyclonal anti-ChAT antibody ( PAB14536 , Abnova, Taipei, Taiwan); and another rabbit polyclonal anti-ChAT antibody (AB143, Millipore via Merck).

Techniques: Expressing, Fluorescence, Flow Cytometry, Incubation, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Tegmental atrophy in isolated REM sleep behaviour disorder: Ex vivo MRI–informed in vivo imaging

doi: 10.1101/2025.10.29.685226

Figure Lengend Snippet: (A) Each row corresponds to a brainstem level shown in the sagittal MRI. Columns from left to right show: (1) ex vivo MRI with color-coded nuclei delineations; (2) Klüver-Barrera staining for cyto- and myeloarchitecture; (3) Choline acetyltransferase (ChAT) immunohistochemistry (corresponding to the black rectangle in (2)); (4) Corticotropin-releasing hormone-binding protein (CRHBP) immunohistochemistry. The areas in (3) and (4) correspond to the black rectangle in (2). Arrows indicate immunoreactive neurons. CRHBP-positive neurons, a marker for REM sleep-on neurons, were localized to the LDTgV. (B) Quantitative cell density analysis of ChAT- and CRHBP-immunoreactive neurons in the PTg, LDTgV, and SubC. Cell densities were calculated from three postmortem human specimens (n = 3 brains). The density of CRHBP-positive neurons was markedly higher in the LDTgV compared to the other nuclei. Values are presented as mean ± standard error of the mean (SEM). Statistical comparisons were made using a one-way ANOVA followed by Tukey’s post-hoc test. For CRHBP, significant differences were found between PTg and LDTgV (* p < 0.05) and between LDTgV and SubC (** p < 0.01). ChAT-positive neurons were distributed across all three nuclei without significant differences. Abbreviations: LDTgV, Laterodorsal tegmental nucleus, ventral part; PTg, Pedunculopontine tegmental nucleus; SubC, Subcoeruleus nucleus

Article Snippet: We used a goat polyclonal anti-human choline acetyltransferase (ChAT) antibody (1:50; E. coli–derived recombinant human ChAT isoform R [Ala2–Pro630], AF3447, R&D Systems, Minneapolis, MN, USA) as a marker for cholinergic neurons, and a rabbit polyclonal anti-human CRHBP antibody (1:100; human CRHBP amino acids 98-136, consistent with previous research ) as a marker for REM sleep-regulating neurons.

Techniques: Ex Vivo, Staining, Immunohistochemistry, Binding Assay, Marker

Journal: Cell

Article Title: Enteric Nervous System-Derived IL-18 Orchestrates Mucosal Barrier Immunity

doi: 10.1016/j.cell.2019.12.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Choline Acetyltransferase/ChAT , Novus Biologicals , NBP2-46620.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Staining, Gene Expression, Sequencing, Software