Journal: Journal of Alzheimer's Disease Reports

Article Title: Identifying Alzheimer's disease genes in apolipoprotein E −/− mice brains with confirmed Porphyromonas gingivalis entry

doi: 10.1177/25424823251332874

Figure Lengend Snippet: Differential expression of genes induced by P. gingivalis FDC 381 (Pg) entry in the ApoE−/− mice versus the sham control groups in the cerebral region of the brain.

Article Snippet: butyrylcholinesterase , Bche-Mm00515326_m1 , Enzyme , No change.

Techniques: Quantitative Proteomics, Control, Binding Assay, Membrane, Activity Assay, Glycoproteomics, Protein Binding, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Formation of PRiMA-linked AChE-BChE G4 hybrid molecules in transfected HEK293T cells. A, HEK293T cells were transfected with equal amounts of cDNAs encoding AChET and BChET for 2 days. Equal amounts of protein from cell lysates were subjected to sucrose density gradient analysis. The enzymatic activities were plotted as a function of the S value, estimated from the position of the sedimentation markers. B, G2 fractions shaded in A were collected for immunoprecipitation by either anti-AChE antibody or anti-BChE antibody. The enzymatic activities of the immobilized AChE and BChE on the beads were determined. The lack of co-immunoprecipitation indicates that only homodimers were produced. C, HEK293T cells were transfected with equal amounts of cDNAs encoding AChET and BChET together with cDNA encoding PRiMA I-HA for 2 days. The amount of PRiMA I-HA cDNA was doubled in the triple transfection to ensure that the ratio of PRiMA to AChE-BChE was the same as in the double transfection. Equal amounts of protein from cell lysates were subjected to sucrose density gradient analysis to analyze the molecular forms, as in A. D, G4 fractions shaded in C were collected for immunoprecipitation as in B. In contrast with B, the co-immunoprecipitation of AChE and BChE indicates the existence of G4 hybrid (mixed) tetramers. The enzymatic activities are expressed in arbitrary units. The values are the means ± S.E., each with triplicate samples (n = 3). Representative gradient profiles and gels are shown (n = 4). Ab, antibody; IP, immunoprecipitation.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Transfection, Sedimentation, Immunoprecipitation, Produced

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Analysis of AChE and BChE oligomers by nondenaturing gel electrophoresis: existence of a single species of PRiMA I-linked AChE-BChE G4 hybrid tetramer. A, G2-enriched fractions from cells expressing AChET, BChET, or AChET + BChET. B, G4-enriched fractions from cells expressing AChET, BChET, or AChET + BChET, together with PRiMA I-HA. The enzymatic activities of AChE and BChE were revealed by Karnovsky staining using acetylthiocholine iodide with ethopropazine (BChE inhibitor) and BTCh with BW284c51 (AChE inhibitor), respectively. BChE oligomers migrate markedly more slowly than the corresponding AChE oligomers; a single intermediate species, possessing both AChE and BChE activities, was observed in G4 fractions from cells expressing both AChET and BChET with PRiMA. C, G4-enriched fractions from cells expressing AChET + BChET + PRiMA I-HA were depleted by anti-HA antibody (from left to right, negative control, 1:12,500, 1:2,500, 1:500, and 1:100 dilutions). Different G4 enzymes in the supernatant were visualized by Karnovsky staining after nondenaturing gel electrophoresis. The decrease of G4 AChE, G4 BChE, and G4 hybrid after the depletion with anti-HA antibody indicates that they are PRiMA-linked. D, gel extracts from G4 AChE, G4 BChE, and G4 hybrid bands in the AChET + BChET + PRiMA I-HA overexpressed cells were analyzed by Western blotting and probed with an anti-HA antibody. Representative gels are shown (n = 3). Ab, antibody; IB, immunoblot.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Nucleic Acid Electrophoresis, Expressing, Staining, Negative Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Formation of AChE-BChE G4 hybrid tetramer in the presence of PRiMA II and QN-GPI. HEK293T cells were transfected with cDNAs encoding AChET and/or BChET with PRiMA (A) or QN-GPI (D) (14) for 2 days. Sucrose density gradient analysis was performed as in Fig. 1A. B and E, G4-enriched fractions shaded in A and D were collected for immunoprecipitation by either anti-AChE or anti-BChE antibody as in Fig. 1B. The co-immunoprecipitation of AChE and BChE indicates the existence of G4 hybrid tetramers. The enzymatic activities are expressed in arbitrary units. The values are the means ± S.E. (n = 3), each with triplicate samples. C and F, G4-enriched fractions shaded in A and D were analyzed by nondenaturing electrophoresis. The enzymatic activities of AChE and BChE were visualized by Karnovsky staining. A new oligomer migrating between G4 AChE and G4 BChE was found in the triple transfection, suggesting that PRiMA II and QN-GPI, like PRiMA I, induce the formation of the AChE-BChE hybrid tetramer. Ab, antibody; IP, immunoprecipitation.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Transfection, Immunoprecipitation, Electrophoresis, Staining

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Analysis of disulfide bonds between subunits of G4 AChE and G4 BChE by nonreducing electrophoresis and Western blotting. A, AChET subunits and PRiMA I-HA were co-expressed in HEK293T cells. G2 and G4 fractions (shaded) from cell lysates were collected following sedimentation in sucrose gradients. Disulfide linkages between AChET subunits and between AChET subunits and PRiMA were analyzed by nonreducing electrophoresis and Western blotting with anti-AChE and anti-HA antibodies, as indicated. The G2 fractions produced a single dimeric band, whereas the G4 fractions produced a dimeric band that did not include PRiMA (light dimer), as well as a dimeric band associated with PRiMA (heavy dimer) and a heavier component, probably representing an AChE tetramer in which all four subunits are disulfide-linked with PRiMA. B, G2 and G4 fractions of AChE were treated with or without peptide N-glycosidase F and analyzed by nonreducing electrophoresis and Western blotting with anti-AChE antibody. AChEs in all different oligomers were N-glycosylated, because of the apparent mass decrease after deglycosylation. The mass of the light dimers in the G4 AChE fraction appeared the same as that in the G2 fractions after the deglycosylation, as indicated by the dotted lines. C, BChET subunits and PRiMA I-HA were co-expressed in HEK293T cells. Disulfide linkages between BChET subunits and between BChET subunits and PRiMA in the G2 and G4 fractions were analyzed by nonreducing electrophoresis and Western blotting with anti-BChE and anti-HA antibodies (lower panel). D, G2 and G4 fractions of BChE were treated with or without peptide N-glycosidase F and analyzed by nonreducing electrophoresis and Western blotting with anti-BChE antibody. The details of the analyses were as for B. Representative gradient profiles and gels are shown, n = 4. Ab, antibody; IB, immunoblot; PNGase F, peptide N-glycosidase F.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Electrophoresis, Western Blot, Sedimentation, Produced

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Oligomerization of human AChE, BChE, and mutants lacking the C-terminal t-peptide or containing exchanged t-peptides. A, comparison of the amino acid sequences of C-terminal t-peptides from human AChE and BChE. Identical residues are indicated by asterisks. Highly and moderately similar residues are indicated by double dots and single dots, respectively. B, schematic representations of AChET, BChET, and their mutants. AChET and BChET are wild-type human AChE and BChE, respectively. AChEΔT and BChEΔT are mutants in which the t-peptides were deleted. AChEBChE-T and BChEAChE-T are chimeras in which the t-peptides of AChE and BChE were exchanged. C, HEK293T cells were transfected with cDNAs encoding AChET, AChEΔT, AChEBChE-T, BChET, BChEΔT, or BChEAChE-T with and without cDNA encoding PRiMA for 2 days. Cell extracts containing equal amounts of enzymatic activity were subjected to sucrose density gradient analysis as in Fig. 1A. The positions of the peaks corresponding to the G1, G2, and G4a forms of AChE (left panel) and BChE (right panel) are shown by vertical dashed lines. Enzymatic activities are expressed in arbitrary units, and one representative result is shown (n = 4).

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Comparison, Transfection, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: AChE and BChE subunits do not form hybrid dimers, even when they contain the same t-peptide. A and D, HEK293T cells were single or double transfected with cDNAs encoding AChE and BChE catalytic subunits, which contained the same t-peptides (AChET and BChEAChE-T or AChEBChE-T and BChET). Sucrose density gradient analysis was performed as in Fig. 1A. B and E, G2 fractions from single and double transfected cells (shaded in A and D) were immunoprecipitated by either anti-AChE or anti-BChE antibodies and absorbed on protein G beads. The enzymatic activities of AChE and BChE immobilized on the beads were determined. C and F, G2 fractions from single and double transfected cell lysates (shaded in A and D) were analyzed by nondenaturing electrophoresis coupled with Karnovsky staining as in Fig. 2. The absence of hybrid dimer in the double transfection indicates that AChE and BChE subunits do not associate, even when containing the same t-peptide. The enzymatic activities are expressed in arbitrary units. The values are the means ± S.E., each with triplicate samples (n = 3). Representative gradient profiles and gels are shown (n = 4). Ab, antibody; IP, immunoprecipitation.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Transfection, Immunoprecipitation, Electrophoresis, Staining

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Formation of G4 hybrids by AChE and BChE subunits containing the same t-peptide. A and D, HEK293T cells were single or double transfected with cDNAs encoding AChE and BChE catalytic subunits, which contained the same t-peptides (AChET and BChEAChE-T or AChEBChE-T and BChET) together with cDNA encoding PRiMA I. The amount of PRiMA I cDNA was doubled in the triple transfection. Sucrose density gradient analysis was performed as in Fig. 1A. B and E, G4 fractions from single and double transfected cells (shaded in A and D) were immunoprecipitated by either anti-AChE or anti-BChE antibody and absorbed on protein G beads; the retained AChE and BChE activities were determined by Ellman assays. C and F, analysis of G4 fractions from double and triple transfected cells by nondenaturing electrophoresis coupled with Karnovsky staining as in Fig. 2. The presence of a new oligomer migrating between G4 AChE and G4 BChE indicates that AChE and BChE, possessing the same t-peptides, can form a PRiMA-linked G4 hybrid. The enzymatic activities are expressed in arbitrary units. The values are the means ± S.E., each with triplicate samples (n = 3). Representative gradient profiles and gels are shown (n = 4). Ab, antibody; IP, immunoprecipitation.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Transfection, Immunoprecipitation, Electrophoresis, Staining

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Existence of PRiMA-linked G4 AChE-BChE hybrid tetramers in chicken brain. A, sedimentation profiles of AChE and BChE molecular forms in extracts from adult chicken brain, as analyzed in Fig. 1A. B, G2 fractions and G4 fractions shaded in A were immunoprecipitated with anti-chicken AChE antibody (3D10) or anti-chicken BChE antibody (7D11) and absorbed onto protein G beads. AChE and BChE activities were determined by Ellman assays. C, immunodepletion of AChE-BChE hybrid molecules with 3D10 antibody. Samples of chicken brain extracts were incubated with or without 3D10 antibody and absorbed onto protein G-agarose beads, and the supernatant was analyzed by sedimentation in sucrose density gradients, as in Fig. 1A. The observed decrease of BChE activity after immunoprecipitation by 3D10 antibody showed that in BChE tetramers, ∼15% of BChE subunits were included in AChE-BChE hybrid tetramers (lower panel). The enzymatic activities are expressed in arbitrary units. The values are the means ± S.E., each with triplicate samples (n = 3). Representative gradient profiles are shown (n = 3). IP, immunoprecipitation.

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Sedimentation, Immunoprecipitation, Immunodepletion, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: The PRiMA-linked Cholinesterase Tetramers Are Assembled from Homodimers

doi: 10.1074/jbc.M110.113647

Figure Lengend Snippet: Developmental profile of PRiMA-linked G4 AChE-BChE hybrid in chicken brain. A, AChE and BChE activities were determined in protein extracts from chicken brain of embryonic (E), postnatal (P), and adult stages, all containing equal amounts of protein. The data were normalized to the basal level of E9. B and C, the level of PRiMA-linked AChE-BChE G4 hybrid molecule was quantified by ELISA. Extracts with equal amounts of AChE activity from chicken brain at different developmental stages were loaded onto ELISA plates precoated with a saturating amount of anti-chicken AChE antibody and incubated for 4 h. B and C, the immobilized AChE activity (B) and BChE activity (C) were determined directly in the ELISA plate after washing. The enzymatic activity was expressed in arbitrary units. The lower panel in C showed the quantitative data of the BChE activity on the hybrid molecules obtained when the AChE activity of input was 100 arbitrary units. The data were normalized by the basal level of E9. All of the values are the means ± S.E., each with triplicate samples (n = 3).

Article Snippet: Immunoprecipitation of AChE and BChE Five hundred μl of G 2 or G 4 fractions, obtained from the sucrose density gradients, were incubated with anti-mammalian AChE antibody E-19 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mammalian BChE antibody N-15 (1:200; Santa Cruz Biotechnology), or anti-chicken AChE monoclonal antibody (3D10, 1:200, purified at 2 μg/ml) ( 15 ) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation

Journal: Scientific Reports

Article Title: Comprehensive proteomic profiling of plasma-derived Extracellular Vesicles from dementia with Lewy Bodies patients

doi: 10.1038/s41598-019-49668-y

Figure Lengend Snippet: Similarities and differences between cohorts from Set 2. ( A ) Correlation multi-scatter plots gave rise to a Pearson correlation of R = 0.75 ± 0.09 for healthy controls and 0.87 ± 0.04 for DLB samples . (B ) ( i ) Comparative protein content analysis of both cohorts by PCA showing components 1 and 2, which account for 35.8% and 19.7%, respectively; ( ii ) Butyrylcholinesterase (BCHE) is one of the proteins differentially identified in the two cohorts based on component 1. (C) Comparison of protein expression by hierarchical clustering analysis with a heat map of the 201 proteins identified in Set 2.

Article Snippet: Pre-coated plates from ELISA kits for gelsolin (GSN; EKU04357) and for butyrylcholinesterase (BCHE; EKU02818) from Biomatik (Cambridge, Ontario, USA), were used following the manufacturer’s instructions.

Techniques: Comparison, Expressing

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) Targeting strategy for the expression of engineered hBChE. The targeting vector contains two Rosa26 homology arms, flanking the expression cassette for hBChE and a selection marker (puromycin resistant gene, Puro) driven by a constitutive promoter UbiC (Ubiquitin C promoter). hBChE and Puro are separated by a self-cleavable peptide T2A. gRNA: guide RNA. (B) Integration of the targeting vector into Rosa26 locus is verified by PCR (left panel) and southern blotting (right panel). Positive clones display an additional band of the expected size. Three independent experiments were performed with similar results. (C) Confirmation of hBChE expression in targeted cells by immunoblots with different antibodies. Numbers on left side indicate molecular weight markers. kD: kilodalton. Full scans of the western blots at . Three independent experiments were performed with similar results. (D) Confirmation of secretion of engineered hBChE in the culture media by ELISA. The box plot indicates the mean (solid diamond within the box), 25th percentile (bottom line of the box), median (middle line of the box), 75th percentile (top line of the box), 5th and 95th percentile (whiskers), 1st and 99th percentile (solid triangles) and minimum and maximum measurements (solid squares). N=number of independent experiments. (E) Cocaine hydrolysis activity in vitro . Cell culture supernatants were collected from cells targeted by hBChE or mBChE. Cocaine hydrolysis activity was examined by a clearance assay in vitro . N=3 (independent experiments), error bars represent standard deviation, and the measure of center represents the average. (F) Cell cycle profiles. FACS (fluorescence activated cell sorting) of control (Ctrl) and hBChE- expressing epidermal stem cells. PI: propidium iodine. Three independent experiments were performed with similar results. Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay, Activity Assay, In Vitro, Cell Culture, Standard Deviation, Fluorescence, FACS, Control

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) Skin organoids are developed from control or hBChE -producing cells, and transplanted to the host mice. Cells were infected with lentivirus encoding firefly luciferase before engraftment to allow intravital imaging of the skin grafts. (B) Histological examination of grafted skin collected from mice grafted with control (GWT) or hBChE skin organoids (GhBChE). Scale bar=50 μm. (C) Sections of grafted skin were immunostained with different antibodies as indicated (Krt10: keratin 10, a marker for early epidermal differentiation, Lor: Loricrin, a marker for early epidermal differentiation, β4: β4-integrin, CD104, a marker for skin basement membrane). Dashed lines denote the basement of skin. Epi: epidermis, Der: dermis. Scale bar=50 μm. (D) Mice are grafted with control or hBChE skin organoids. Presence of hBChE in blood was determined by ELISA for 10 weeks after engraftment (n=5 mice in each group). (E) Cocaine pharmacokinetics in the NAc (nucleus accumbens) after an i.p. administration of 10 mg/kg cocaine in GhBChE and GWT mice (n = 6 for each group). Data are plotted as mean±SEM (standard error of the mean). A two-compartment in vivo pharmacokinetic model was built to represent the cocaine concentration-time profile in the NAc. (F) Changes in DA (dopamine) level in the NAc after an i.p. administration of 10 mg/kg cocaine in GhBChE and GWT mice (n = 6 for each group). Individual lines for each animal were plotted. Treatment×time interaction: F 6, 70 =6.549, P＜0.0001, two-way ANOVA. (G-H) Cocaine-induced locomotor activity in in GhBChE and GWT mice (n = 11 for GWT, n=8 for GhBChE). Non-linear dose versus response curve was simulated to represent cocaine-induced dose-response locomotor activity. (G) Total distance traveled after 0, 10, 20, 40 mg/kg cocaine i.p. injection. Treatment×doses interaction: F 3, 68 =11.83, P＜ 0.0001, two-way ANOVA. (H) Stereotypic counts after 0, 10, 20, 40 mg/kg cocaine i.p. injection. Treatment×doses interaction: F 3, 65 =6.223, P=0.0009, two-way ANOVA. (I) Lethality rates after injection of 40, 80, 120, 160 mg/kg cocaine in GhBChE and GWT mice (n=3 independent experiments, for each experiment, 8 animals were examined in each test group). Individual data point represents the result from each experiment. Error bars represent SEM, and the measure of center represents the average. Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Control, Infection, Luciferase, Imaging, Marker, Membrane, Enzyme-linked Immunosorbent Assay, Drug discovery, In Vivo, Concentration Assay, Activity Assay, Injection

Journal: Nature biomedical engineering

Article Title: Genome-edited skin epidermal stem cells protect mice from cocaine-seeking behaviour and cocaine overdose

doi: 10.1038/s41551-018-0293-z

Figure Lengend Snippet: (A) The AAVS1 targeting strategy for expression of engineered hBChE. The targeting vector contains two AAVS1 homology arms, flanking the expression cassette for hBChE and a selection marker (puromycin resistant gene, Puro) driven by a constitutive promoter UbiC (Ubiquitin C promoter). hBChE and Puro are separated by a self-cleavable peptide T2A. (B) Integration of the targeting vector into AAVS1 locus is verified by southern blotting. Positive clones display an additional band of the expected size. Three independent experiments were performed with similar results. (C) Expression of hBChE is confirmed in targeted cells by immunoblots with different antibodies as indicated. Full scans of the western blots at . Three independent experiments were performed with similar results. (D) Secretion of engineered hBChE in the culture media is confirmed by ELISA. N=3 (independent experiments). Box plots were generated with the same elements as described in . (E) Image of nude mouse grafted with organotypic human skin culture. Intravital imaging shows efficient incorporation of grafted cells expressing luciferase (right side) or control cells (left side) upon engraftment. (F) Sections of grafted skin were immunostained with different antibodies as indicated. Dashed lines denote the basement of skin. Scale bar=50 μm. (G) Mice are grafted with control or hBChE skin organoids. Presence of hBChE in blood was determined by ELISA for 8 weeks after engraftment (n=3 mice in each group). Text in this legend has been adapted from our previous work in ref. .

Article Snippet: Human BChE quantikine ELISA kit was obtained from R&D systems (Minneapolis, MN).

Techniques: Expressing, Plasmid Preparation, Selection, Marker, Ubiquitin Proteomics, Southern Blot, Clone Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Imaging, Luciferase, Control

Journal: bioRxiv

Article Title: Gut microbiota gate host exposure to metabolites from dietary Solanums

doi: 10.1101/2024.03.20.584512

Figure Lengend Snippet: A) Total steroidal alkaloid concentration in the serum of C57BL/6J mice fed potato-supplemented diet and treated with antibiotics. Aglycone concentrations were estimated using a solanidine standard curve. Data shown are the mean±SD (n=5 mice). P values were determined using a two-sided Welch’s T test. B) In vitro inhibition of recombinant human AChE activity by potato steroidal alkaloids. Data shown are normalized to AChE treated with vehicle only (DMSO for solanine, chaconine and ethanol for solanidine) with a total vehicle concentration of 1% for all treatments. Values shown are the mean±SD (n=3 assays per concentration). 5 ng of AChE was used in each reaction. C) In vitro inhibition of cholinesterase activity by 50 μM potato SAs in human protein homogenates from normal colonic tissue (n=3 donors) and Caco-2 colonic epithelial cells. Activity was normalized to the activity of the vehicle-treated sample. Values shown are the mean±SD of three replicate assays per homogenate. 5 ng each of recombinant human AChE and BChE were used for pure protein reactions, while 35 μg total protein was used for each homogenate. Multiplicity-adjusted P values were calculated using Tukey’s multiple comparison tests. D) Inhibition of recombinant human AChE activity by spent media from human stool (n=11 donors) incubated with 20 μM solanine in SAAC minimal medium. Activity was normalized to the activity of AChE treated with spent media from the same donor sample incubated with vehicle only. Values shown are the mean±SD of three AChE activity assays for each donor and compound combination. The relative abundances of select metabolites following incubation with solanine are shown below for each stool sample. Values shown are EIC peak areas normalized to the maximum peak area across all samples. P values were determined using a two-sided Welch’s T test. Ns: not significant with P>0.05.

Article Snippet: BChE activity was analogously measured using 5 ng of recombinant human BChE (R&D Systems) and butyrylthiocholine iodide.

Techniques: Concentration Assay, In Vitro, Inhibition, Recombinant, Activity Assay, Comparison, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage

doi: 10.1167/iovs.65.12.37

Figure Lengend Snippet: Induction of retinal organoids from NMNAT1-KO hiPSC. ( A ) Bright field photographs of the retinal organoids made from control or NMNAT1-KO hiPSCs were taken at the cultured days 7, 11, 14, and 21, and week 8. Representative photographs from 10 to 20 colonies of few independent cultures are shown. ( B, C ) Colonies at day 15 ( B ) and 21 ( C ) were immunostained with anti-TUBB3 antibody, and nuclei were visualized by DAPI staining. In B , the red square regions of upper panels are enlarged in the lower panels . Representative photographs of six samples from two independent cultures are shown. ( D, E ) Colonies at day 63 were frozen sectioned, and the sections were immunostained with anti-Ki67 and -BRN3B antibodies ( D ), or anti-CRX or -RXRG antibodies ( E ). Representative photographs of three sections from three independent organoids of two independent cultures are shown. Nuclei were visualized by staining with DAPI ( B – E ). Scale bars are 500 µm ( A, B, C ) and 100 µm ( D, E ).

Article Snippet: Immunohistochemistry of frozen sections was done as described previously., Primary antibodies used were mouse monoclonal antibodies against Ki67 (BD Biosciences, #55060924), TUBB3 (R&D Systems, MAB1195), goat polyclonal antibody against human BRN3B (Santa Cruz, sc-60267), CRX (Abnova Corp., 4G11, H00001406-M02), RXRG (Santa Cruz, A-2, sc-365252), and chick polyclonal antibody against GFP (Abcam, ab13970).

Techniques: Control, Cell Culture, Staining