cht1  (Danaher Inc)

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 1 Radioactive choline uptake assay of human CHT1 expressed in HEK293 cells. a Radioactive choline uptake of the CHT1-expressing HEK293 cells (wild-type, blue bar) as compared to the background radioactivity from the control cells transfected with the empty vector (EV, gray bar) 10 min after adding 0.1 mM choline with 10% of [3H]-choline. The inhibition of CHT1-mediated choline uptake was measured at 0.1 mM HC3 or 1 mM ML352. Data are mean ± SEM (n = 3 independent experiments). One-way ANOVA; ****P ≤0.0001. b Concentration- dependent choline uptake. Data points are mean ± SEM (n = 3 independent experiments) and fitted to the Michaelis–Menten equation with KM = 3.86 ± 0.68 mM. c Na+ and Cl–-dependent choline uptake. Data are mean ± SEM (n = 4 independent experiments) and are normalized against the radioactivity measurement with NaCl in the reaction solution. One-way ANOVA; ****P ≤0.0001. d, e Concentration-dependent inhibition of CHT1-mediated choline uptake by HC3 (d) and ML352 (e). Data points are mean ± SEM (n = 3 for HC3 and n = 3–6 for ML352) and fitted to the three-parameter dose–response curves (GraphPad Prism 9) with IC50 of 4.98 ± 1.04 nM for HC3 and 168.6 ± 49.4 nM for ML352.

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques: Expressing, Radioactivity, Control, Transfection, Plasmid Preparation, Inhibition, Concentration Assay

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 2 The overall structure of the apo human CHT1 in inward-open conformation. a Topology diagram of human CHT1. Segments of TM0, TMs 1–5, TMs 6–10, and TMs 11–12 are individually colored. b Side view of 3D reconstruction (left) and cartoon representation of the CHT1apo structure with the four segments individually colored as the topology diagram in a. c Bottom view of the apo CHT1 from the intracellular side. The red dashed oval marks the entrance of the intracellular vestibule. d Side view of the cross-section of the surface-rendered CHT1apo

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques:

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 3 The substrate-bound CHT1Chol structure. a Bottom view of the CHT1Chol structure with the bound ions and choline highlighted in red dashed boxes. b Zoomed-in view of choline-binding in CHT1. The density (blue mesh) for choline is contoured at 6 σ. Key choline-interacting residues are shown in sticks. The bound Cl– and Na+ are also shown for reference. c The effect of mutagenesis at the substrate-binding site on choline uptake. Data are mean ± SEM (n = 4–6 independent experiments) and are normalized against the measurement from the wild-type CHT1. One-way ANOVA; ****P ≤0.0001. d Zoomed-in view of Cl– binding in CHT1. The density (blue mesh) for the Cl– ion is contoured at 4 σ. Key Cl–-interacting residues are shown in sticks. The dotted lines mark the coordination between the Cl– ion and the protein atoms. The inset provides an alternative view of the 63VGGGY67 region for enhanced clarity. e Zoomed-in view of Na+ binding at Na2 in CHT1. The density (blue mesh) for the Na+ ion is contoured at 4 σ. Key Na+ -interacting residues are shown in sticks. The dotted lines mark the coordination between the Na+ ion and the protein atoms. The surrounding residues for the Na3 site (red dotted circle) are also shown. f The effect of mutagenesis at Na2 and Na3 sites on choline uptake. Data are mean ± SEM (n = 3–8 independent experiments) and are normalized against the measurement from the wild-type CHT1. One-way ANOVA; ****P ≤0.0001.

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques: Binding Assay, Mutagenesis

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 5 The structure of HC3-inhibited CHT1 in outward-open conformation. a Side view of the cross-section of the surface-rendered CHT1HC

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques:

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 6 The structure of ML352-inhibited CHT1 in inward-open conformation. a The structure of CHT1ML with the bound ML352 inhibitor on the external surface of the transporter. The zoomed-in view of the surface-rendered CHT1ML illustrates the space-filling binding of ML352. Cryo-EM density of ML352 in the contour level of 0.42 in ChimeraX. b Structural comparison between CHT1apo and CHT1ML illustrates subtle structural change at the EL6 loop between the two. c Zoomed-in view of the protein–inhibitor interactions.

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques: Binding Assay, Cryo-EM Sample Prep, Comparison

Journal: Cell discovery

Article Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.

doi: 10.1038/s41421-024-00731-7

Figure Lengend Snippet: Fig. 7 A working model for the Na+ and Cl–-dependent choline transport in CHT1. The orange arrows mark the conformational changes driven by the choline-binding from the extracellular side.

Article Snippet: The following primary antibodies were used for probing corresponding proteins: anti-Strep-tag mouse monoclonal antibody (IBA Lifesciences, cat# 2-1507-001, 1:10,000 dilution) for wild-type CHT1 and mutants; anti-Na+/ K+-ATPase subunit alpha-1 rabbit monoclonal antibody (Abcam, cat# ab76020, 1:10,000) for plasma membrane marker; anti-beta Actin mouse monoclonal antibody (Santa Cruz, cat# sc-69879, 1:2000 dilution) for cytosolic marker.

Techniques: Binding Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: Electroacupuncture regulates Rab5a‐mediating NGF transduction to improve learning and memory ability in the early stage of AD mice

doi: 10.1111/cns.14743

Figure Lengend Snippet: The expression levels of cholinergic‐related proteins in the basal forebrain and hippocampus. (A–E) The expression levels of vAchT (A), ChT1 (B), AchE (C), m1AchR (D), and m2AchR (E) in the basal forebrain after EA intervention. (F‐J) The expression levels of vAchT (F), ChT1 (G), AchE (H), m1AchR (I), and m2AchR (J) in the hippocampus after EA intervention ( n = 6 for each group, ** p < 0.01/*** p < 0.001 vs WT group, # p < 0.05/## p < 0.01/### p < 0.001 vs AD group, && p < 0.01 vs EA group). (K) Greyscale image of proteins. vAchT, vesicular acetylcholine transporter; ChT1, Choline transporter; AchE, enzyme acetylcholinesterase; m1AchR, Type‐1 muscarinic Acetylcholine receptor; m2AchR, Type‐2 muscarinic Acetylcholine receptor.

Article Snippet: The membranes were incubated with primary antibodies at 4°C: Rab5a (1:1000; 24 h, CST, E6N8S); Rabep1 (1:5000; 24 h, Abcam, ab176578); TrkA (1:300; 36 h, Abcam, ab216626); pTrkA (1:500; 36 h, Invitrogen, PA5‐37672); AKT (1:1000; 24 h, CST, #4691); pAKT (1:1000; 36 h, CST, #4060); ERK (1:5000; 24 h, Abcam, ab184699); pERK (1:2000; 24 h, CST, #4370); ChAT (1:5000; 24 h, Abcam, ab181023); AchE (1:5000; 36 h, Abcam, ab183591); vAchT (1:1000; 36 h, Sigma, sab4200559); ChT1 (1:5000; 24 h, Abcam, ab154186); m1AchR (1:1000; 36 h, boster, BA1543); m2AchR (1:5000; 24 h, Abcam, ab109226); GAPDH (1:5000; 24 h, Proteintech, 60,004–1‐1 g); and β‐actin (1:5000; 24 h, Proteintech, 66,009–1‐1 g).

Techniques: Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: CircFBXW4 Suppresses Colorectal Cancer Progression by Regulating the MiR-338-5p/SLC5A7 Axis.

doi: 10.1002/advs.202300129

Figure Lengend Snippet: Figure 7. CircFBXW4 regulates SLC5A7 expression by acting as a sponge for miR-338-5p. A) Relative expression of candidate mRNAs in SW480 cells transfected with the miR-338-5p mimic. B) Relative expression of candidate mRNAs in SW620 cells transfected with the miR-338-5p inhibitor. C) Relative protein levels of SLC5A7 in CRC cells transfected with the miR-338-5p mimic or inhibitor. D) The relative expression of SLC5A7 in CRC tissues and matched adjacent normal tissues was determined by qRT‒PCR (n = 40). E) Pearson correlation analysis between the expression levels of miR-338-5p and SLC5A7 in our own patient cohort (n = 40). F) Schematic illustration of the SLC5A7-WT and SLC5A7-MUT luciferase reporter vectors. G) Relative luciferase activity was measured in 293T cells after cotransfection with SLC5A7-WT or SLC5A7-MUT and the miR-338-5p mimic or NC. H) The relative mRNA and protein expression levels of SLC5A7 were measured by qRT‒PCR and western blotting in SW480 cells transfected with si-NC or si-circFBXW4 with or without the miR-338-5p inhibitor. I) The relative mRNA and protein levels of SLC5A7 were measured by qRT‒PCR and western blotting in SW620 cells transfected with vector or the circFBXW4-OE plasmid with or without the miR-338-5p mimic. The data are shown as the means ± SDs; *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The primary antibodies used to detect Ki-67, PCNA, and Caspase3 were purchased from ABclonal (China), and the primary antibody used to detect SLC5A7 was purchased from ProteinTech (China).

Techniques: Expressing, Transfection, Luciferase, Activity Assay, Cotransfection, Western Blot, Plasmid Preparation