Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of increasing cholesterol (CHS) concentrations on OCTN1 mediated transport of TEA and acetylcholine. The recombinant OCTN1 was reconstituted in liposomes as described in . except that ( a and b ) proteoliposomes batches used for each time course experiment were reconstituted in the presence of 0 (○), 2.1 (●), 4.2 (□), 8.3 (■) % CHS (corresponding to 0, 21, 42 and 83 µg CHS/mg total lipids, respectively). ( a ) Transport was started adding 0.1 mM [ 14 C]-TEA ( b ) or 0.1 mM [ 3 H]-acetylcholine, at time zero to proteoliposomes and stopped at the indicated times as described in . Time course data were interpolated in a first order rate equation. The values are means ± SD from three different experiments. ( c ) Transport rates of [ 14 C]-TEA (○) or [ 3 H]-acetylcholine (●) uptake, calculated as K x transport at equilibrium from ( a , b ), reported as function of the % CHS.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of CHS on regulation of OCTN1 by ATP or NaCl. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the presence of 8.3% CHS corresponding to 83 µg CHS/mg total lipids and ( a ) in the presence of the indicated ATP concentrations. Transport was started adding 0.1 mM [ 14 C]-TEA (white column, ○) or [ 3 H]-acetylcholine (grey column, □) at time zero to proteoliposomes. ( b ) Increasing NaCl extraliposomal concentration were added together to the radioactive substrates. The transport reaction was stopped at 20 min. Percent residual activity with respect to the control data were interpolated in an IC 50 equation (Dose-response curves). The values are means ± SD from three experiments. Significantly different for * p < 0.01 as calculated from Student’s t test analysis.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes, Concentration Assay, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of hemicholinium and TEBA on the OCTN1 activity measured in the presence or absence of CHS. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the absence (○) or in the presence (●) of 8.3% CHS corresponding to 83 µg CHS/mg total lipids. ( a ) Transport was started adding 0.1 mM [ 3 H]-acetylcholine at time zero to proteoliposomes in the presence of increasing external hemicholinium concentrations (0-0.1-0.3-0.5-0.8-1-1.5-2.6 mM) or ( b ) adding 0.1 mM [ 14 C]-TEA in the presence of increasing external TEBA concentrations (0-0.5-1-2.5-5-10-20 mM). The transport reaction was stopped at 20 min. Percent residual activity with respect to the control data were interpolated in an IC 50 equation (Dose-response curves). The values are means ± SD from three experiments.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Activity Assay, Recombinant, Liposomes, Control

Journal: International Journal of Molecular Sciences

Article Title: Effect of Cholesterol on the Organic Cation Transporter OCTN1 (SLC22A4)

doi: 10.3390/ijms21031091

Figure Lengend Snippet: Effect of CHS on OCTN1 transport kinetics. The recombinant OCTN1 was reconstituted in liposomes as described in . except that in the absence (○) or in the presence (●) of 8.3% CHS corresponding to 83 µg CHS/mg total lipids. The transport rate was measured adding [ 14 C]-TEA ( a ) or [ 3 H]-acetylcholine ( b ) at the indicated increasing concentrations to proteoliposomes. The transport reaction was stopped at 10 min. Data were plotted according to Lineweaver–Burk as reciprocal transport rate vs reciprocal TEA ( a ) or acetylcholine ( b ) concentration. The values are means ± SD from three experiments.

Article Snippet: Amberlite XAD-4, egg yolk phospholipids (3-sn-phosphatidylcoline 60%), CHS, Triton-X100, Sephadex G-75, TEA and acetylcholine were purchased from Merck Life Science s.r.l., 20149 Milano, Italy; tetraethylammonium bromide [ethyl-1- 14 C] was from ARC (American Radiolabeled Chemicals, St. Louis, MO 63146 USA); acetylcholine iodide [acetyl- 3 H] was from Perkin-Elmer Italia S.p.A. 20126 Milano, MI, Italy.

Techniques: Recombinant, Liposomes, Concentration Assay

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 primary antibodies included rabbit anti-M1-mAChR polyclonal antibody (Biodesign International, Cat. # Q4A173R, Saco, ME, USA), rabbit anti-M2-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-002, Jerusalem, Israel), rabbit anti-M3-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-006, Jerusalem, Israel), mouse anti-M4-mAChR monoclonal antibody (Chemicon International, Cat. # MAB1578, Temecular, CA) and rabbit anti-M5-mAChR antibody (Biodesign International, Cat. # Q4A871R, Saco, ME, USA).

Techniques:

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: Fluorescent labeling of mAChR subtype expression on vestibular peripheral structures in the crista, utricle and ganglion. The results shown here are the anti-M4 antibody reacted with a red fluorescent secondary antibody while other mAChR subtypes reacted with a green secondary antibody. No differences were found when the red/green fluorescent secondary antibodies were exchanged. The co-expression of M4 with M1, M3 and M5 on nerve fibers, nerve terminals and ganglion cells are marked by arrows. Myelin sheaths and Schwann cells surrounding ganglion cells are pointed at by single arrowheads. Cuticular plates w/ or w/o protruding cilia are pointed at by double arrows. The labeling above the row of hair cells in utricle (especially in E2) is the artifact induced by labeling the otoconial membrane which was not removed. Scale bars = 10 μm.

Article Snippet: The primary antibodies included rabbit anti-M1-mAChR polyclonal antibody (Biodesign International, Cat. # Q4A173R, Saco, ME, USA), rabbit anti-M2-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-002, Jerusalem, Israel), rabbit anti-M3-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-006, Jerusalem, Israel), mouse anti-M4-mAChR monoclonal antibody (Chemicon International, Cat. # MAB1578, Temecular, CA) and rabbit anti-M5-mAChR antibody (Biodesign International, Cat. # Q4A871R, Saco, ME, USA).

Techniques: Labeling, Expressing

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: DAB staining of (A) semicircular canal crista and (B) vestibular ganglion. The number in each panel corresponds to the mAChR subtype number. Myelin sheaths and Schwann cells circumscribe the nerve fibers and ganglion cells (black arrows). Nerve fibers expressing M1, M2, M4 and M5 traverse the basement membrane and terminate in the neuroepithelium (arrowheads). There is no M3 expression on peripheral nerve terminals inside of the neuroepithelium. The myelin sheath staining stops just beneath the basement membrane (double arrowheads in A-3). Scale bars = 10 μm.

Article Snippet: The primary antibodies included rabbit anti-M1-mAChR polyclonal antibody (Biodesign International, Cat. # Q4A173R, Saco, ME, USA), rabbit anti-M2-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-002, Jerusalem, Israel), rabbit anti-M3-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-006, Jerusalem, Israel), mouse anti-M4-mAChR monoclonal antibody (Chemicon International, Cat. # MAB1578, Temecular, CA) and rabbit anti-M5-mAChR antibody (Biodesign International, Cat. # Q4A871R, Saco, ME, USA).

Techniques: Staining, Expressing

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: Summary of mAChR subtype expression on the peripheral vestibular neurons

Article Snippet: The primary antibodies included rabbit anti-M1-mAChR polyclonal antibody (Biodesign International, Cat. # Q4A173R, Saco, ME, USA), rabbit anti-M2-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-002, Jerusalem, Israel), rabbit anti-M3-mAChR antibody (Alomone Labs Ltd., Cat. # AMR-006, Jerusalem, Israel), mouse anti-M4-mAChR monoclonal antibody (Chemicon International, Cat. # MAB1578, Temecular, CA) and rabbit anti-M5-mAChR antibody (Biodesign International, Cat. # Q4A871R, Saco, ME, USA).

Techniques: Expressing