Journal:

Article Title: Muscarinic Acetylcholine Receptor Subtype Expression in Avian Vestibular Hair Cells, Nerve Terminals and Ganglion Cells

doi: 10.1016/j.neuroscience.2007.02.019

Figure Lengend Snippet: Antibodies and Antigens Used for Muscarinic Receptor Subtype Studies

Article Snippet: The amino and carboxyl terminals are identified by H 2 N and COOH, respectively. table ft1 table-wrap mode="anchored" t5 caption a7 mAChR Subtypes Item * Cat. # Host Source Company Epitope Identity to Pigeon Pigeon Accession # mAChR M1 Ab Q4A173R Rabbit Biodesign Amino acid --- --- Ag A4A730H Human International 451–460 mAChR M2 Ab AMR-002 Rabbit Alomone Labs Amino acid 87/132 (65.9%) {"type":"entrez-nucleotide","attrs":{"text":"AY838767","term_id":"56681308","term_text":"AY838767"}} AY838767 Ag For AMR-002 Human 225–356 mAChR M3 Ab AMR-006 Rabbit Alomone Labs Amino acid 17/19 (89.5%) {"type":"entrez-nucleotide","attrs":{"text":"AY838768","term_id":"56681310","term_text":"AY838768"}} AY838768 Ag For AMR-006 Rat 461–479 Chemicon Amino acid 101/194 (52.1%) {"type":"entrez-nucleotide","attrs":{"text":"AY838766","term_id":"56681306","term_text":"AY838766"}} AY838766 mAChR M4 Ab MAB1578 Mouse International 217–401 Santa Cruz Amino acid 92/184 (50%) Protein sc-4505 WB Human Biotechnology 220–394 mAChR M5 Ab Q4A871R Rabbit Biodesign Amino acid 13/16 (81.3%) {"type":"entrez-nucleotide","attrs":{"text":"DQ357059","term_id":"86161661","term_text":"DQ357059"}} DQ357059 Ag A4A738H Human International 516–531 Open in a separate window * Ab, Antibody; Ag, Antigen.

Techniques:

Journal: PLoS ONE

Article Title: M1 Muscarinic Receptor Activation Mediates Cell Death in M1-HEK293 Cells

doi: 10.1371/journal.pone.0072011

Figure Lengend Snippet: (A) PCR was conducted to assess M1 expression by vector-transfected (HEK293-Vec) and M1 transfected HEK293 cells (HEK293-M1) using primers for M1 mAChR. The integrity of cDNA samples was confirmed using GAPDH. Samples are as follows; HEK293-Vec cDNA in lane 1, HEK293-Vec -RT/RNA in lane 2, HEK293-M1 cDNA in lane 3, and HEK293-M1–RT/RNA in lane 4. Samples are representative of those used in subsequent functional studies. (B) Immunocytochemical localisation of M1 receptors in HEK293-M1 cells using anti-HA.11 antibody (middle panel) and M1 antibody (right-hand panel). Left panel shows no primary control Image. (C) Analysis of M1 receptor cell-surface expression and internalization by carbachol. Cell surface receptors were live-labeled (see methods) using the HA.11antibody prior to stimulation with water-control or carbachol treatment. The M1 mAChRs were typically localised at the plasma membrane after water treatment, but after 1 h carbachol treatment for the M1 mAChRs were internalised, as shown by the increased punctate cytoplasmic staining (arrows) and reduced staining intensity on the cell surfaces. Scale bar: 50 µm. Data are representative of at least three independent experiments. (D) shows the time-course of M1 receptor internalization after carbachol addition using the granularity assay in Metamorph to measure internalized receptors (as intracellular granules). The graph shows that 5–60 minutes after carbachol addition there is internalization of M1 receptors.

Article Snippet: The sources of primary antibodies and the species they were derived from for the following proteins are as follows – EGR-1 (rabbit, sc-189) from Santa Cruz Biotechnology, M1 mAChR (rabbit, M9808) from Sigma, HA.11 (mouse, MMS-101P) from Covance, ERK1/2 (rabbit, 9102), phospho-p44/42 ERK1/2 (rabbit, 9101) and cleaved caspase-3 (rabbit, 9661L) from Cell Signaling Technologies, pCREB (rabbit, 06-519) from Upstate Biotechnology, β-Actin (mouse, ab6276) from Abcam, and BrdU (mouse, 11 170 376 001) from Roche.

Techniques: Expressing, Plasmid Preparation, Transfection, Functional Assay, Labeling, Staining

Journal: The Journal of Veterinary Medical Science

Article Title: Expression of Adrenergic and Cholinergic Receptors in Murine Renal Intercalated Cells

doi: 10.1292/jvms.14-0315

Figure Lengend Snippet: Expression of mAChRs in M-1 cells and immunohistochemical localization of the m1 ADR in the mouse kidney. A) RT-PCR (upper column) of m1, m3, m4 and m5 receptor subtypes in M-1 total cellular RNA (lane 1, arrowheads). Genomic DNA was used as a control (lane 3). No positive band was observed in RTase (−) negative control lane (lane 2). Immunoblot (lower column) demonstrating the presence of the m1 receptor subtype in the M-1 total membrane protein fraction. The most intense band is observed at 55 to 70 kDa (arrowhead). B) to E) Immunohistochemistry demonstrating the presence of the m1 mAChR (B and D) and AQP6 (C and E) in the cortex (B and C) and renal outer medulla (D and E). Arrows indicate AQP6-positive, m1-positive cells. Arrowheads indicate AQP6-positive, m1-negative cells. Reactions of m1 antibody were stronger to the medullary collecting ducts (B) than to the CCDs (D). Bar=20 µ m.

Article Snippet: Membranes were incubated with diluted primary antibodies including rabbit anti-AQP6 polyclonal antibody (pAb, #AQP-006, Alomone Labs, Jerusalem, Israel), rabbit anti-AQP2 pAb (#AQP-002, Alomone labs), rabbit anti-ADR b2 pAb (#Sc-569, Santa Cruz, TX, U.S.A.) and rabbit anti-mAChR m1 pAb (#010, Alomone Labs) for 2 hr at room temperature or at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Reverse Transcription Polymerase Chain Reaction, Negative Control, Western Blot, Immunohistochemistry

Journal: ACS Omega

Article Title: Ergothioneine Stimulates Ca 2+ -Mediated Brain-Derived Neurotrophic Factor Expression in NE-4C Nerve Cells

doi: 10.1021/acsomega.4c09920

Figure Lengend Snippet: Effect of EGT on BDNF expression and CREB phosphorylation in NE-4C nerve cells. The expression of BDNF (A), ratio of pCREB and CREB (B), and m/n AChR (C) in NE-4C nerve cells treated with 1, 10, and 100 μM EGT were evaluated using Wes analysis. Protein expression of BDNF and m/n AChR was normalized using the electropherogram peak area of the total protein in each lane. The chemiluminescent signal was displayed as a virtual blot-like image, and an electropherogram was generated based on the molecular weight. Values are expressed as the mean ± SD ( n = 3). Statistical analyses were performed using one-way ANOVA followed by Dunnett’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs control; N.S ., no significance at p > 0.05.

Article Snippet: The primary antibodies used for immunoblotting were as follows: BDNF (anti-BDNF antibody, rabbit monoclonal antibody, Lot: 1035294-1, 1:50 dilution, Abcam, Cambridge, UK), phospho-cAMP response element-binding protein (CREB) {pCREB (Ser133) [1B6], mouse monoclonal antibody, Lot: 11, 1:250 dilution, Cell Signaling Technology, Danvers, MA, USA}, CREB (anti-CREB antibody [D76D11], rabbit monoclonal antibody, Lot: 7, 1:50 dilution, Cell Signaling Technology), muscarinic AChR (anti-mAChR M1 antibody, Lot: 822203059, 1:50 dilution; GeneTex, CA, USA), and nicotinic AChR (anti-nAChR α4/CHRNA4 antibody, Lot: GR83312-9, 1:50 dilution; Abcam).

Techniques: Expressing, Phospho-proteomics, Generated, Molecular Weight, Control