Journal: Nature Communications

Article Title: Rapid amide ligation between α-halo acylsilanes and amines under aqueous conditions

doi: 10.1038/s41467-025-65617-y

Figure Lengend Snippet: LC-MS and SDS-PAGE gel analysis of proteins modified. Reaction of lysozyme ( A , B ), chymotrypsinogen A ( C ), myoglobin ( D ) and ribonuclease A ( E ) with α-halo acylsilanes in DMSO/NH 4 OAc (20 mM) = 4/1 at 25 °C for 1 h, independent replicates (n = 4) of SDS-PAGE gel analysis were performed in ( B ), with similar results. The mass spectrometry data had not been deconvoluted, and the number of added ions had been marked in the figure. (+1 mod. = singly modified protein, +2 mod. = doubly modified protein, etc.).

Article Snippet: Ribonuclease A, from bovine pancreas was purchased from MedChemExpress (MCE).

Techniques: Liquid Chromatography with Mass Spectroscopy, SDS Page, Modification, Mass Spectrometry

Journal: Journal of Thoracic Disease

Article Title: Regulatory mechanisms of haptoglobin on particulate matter-induced epithelial-to-mesenchymal transition in bronchial epithelial cells

doi: 10.21037/jtd-2024-2013

Figure Lengend Snippet: Exogenous HP increased the migration ability of BEAS-2B cells and facilitated PM-induced EMT and activation of the STAT3/ERK pathway. (A) BEAS-2B cells were treated with varying doses (0, 10, 100, and 1,000 ng/mL) of rhHP for 24 h. Representative images of cell migration were observed through wound-healing assay. (B) Quantification of the wound healing assay. (C) BEAS-2B cells were treated with 100 ng/mL rhHP and 200 μg/mL PM for 24 h. The protein expression of E-cadherin and vimentin and activation of the STAT3/ERK signaling pathway in cells were analyzed using Western blotting (n=3). (D-G) The optical densities of protein bands. The values are the mean ± SEM. **, P<0.01 vs. control group. # , P<0.05; ## , P<0.01 vs. control group. HP, haptoglobin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PM, particulate matter; EMT, epithelial-to-mesenchymal transition; rhHP, recombinant human HP; SEM, standard error of measurement; STAT3, signal transducer and activator of transcription 3; ERK, extracellular regulated protein kinases.

Article Snippet: Recombinant human HP (rhHP) was obtained from MedChemExpress (MCE; Monmouth Junction, NJ, USA).

Techniques: Migration, Activation Assay, Wound Healing Assay, Expressing, Western Blot, Control, Recombinant

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 6. Cont, control; Wnta1, Wnt agonist 1; U, U73122; the collection time for each fraction is 2 min. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983; p-TAK1, phospho-TAK1; t-TAK1, total-TAK1. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor, an inhibitor of TAK1; p-ATF2, phospho-ATF2; t-ATF2, total-ATF2. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: The WNT/Ca 2+ pathway promotes atrial natriuretic peptide secretion by activating protein kinase C/transforming growth factor-β activated kinase 1/activating transcription factor 2 signaling in isolated beating rat atria

doi: 10.4196/kjpp.2022.26.6.469

Figure Lengend Snippet: Data were expressed as means ± SE. n = 5. Cont, control; Wnta1, Wnt agonist 1; U, U73122; Go, Gö6983, TI, TAK1 inhibitor. *p < 0.05 vs. control, # p < 0.05 vs. Wnta1.

Article Snippet: The phospholipase C antagonist U73122, transforming growth factor-β activated kinase 1 (TAK1) inhibitor 1, and inhibitor of PKC Go 6983 were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Control

Journal: Scientific Reports

Article Title: CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

doi: 10.1038/s41598-020-76449-9

Figure Lengend Snippet: SaCas9 mRNA expression.

Article Snippet: The primary antibody used was Mouse SaCas9 monoclonal antibody [6H4] (EpiGentek, Catalog #A-9001), and the secondary antibody was Goat anti-Mouse IgG H&L [FITC] antibody (Abcam, Catalog #AB6785).

Techniques: Expressing, CRISPR

Journal: Scientific Reports

Article Title: CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

doi: 10.1038/s41598-020-76449-9

Figure Lengend Snippet: Western blot for SaCas9 protein expression. Protein expression for SaCas9 was shown in the positive control as well as in A17L and I2L targets 48 h post-infection. Protein expression was not detected for either targets at 72 h post-infection. (Image is cropped. Full-length blot is presented in Supplementary Fig. ).

Article Snippet: The primary antibody used was Mouse SaCas9 monoclonal antibody [6H4] (EpiGentek, Catalog #A-9001), and the secondary antibody was Goat anti-Mouse IgG H&L [FITC] antibody (Abcam, Catalog #AB6785).

Techniques: Western Blot, Expressing, Positive Control, Infection

Journal: Scientific Reports

Article Title: Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

doi: 10.1038/s41598-019-46357-8

Figure Lengend Snippet: Principle of Bindel-PCR for identifying CRISPR/Cas9-induced biallelic mutants. ( a ) Outline of Bindel-PCR. 1st PCR is carried out under standard concentrations of Mg 2+ (1.5–2.5 mM) for confirming successful amplification of a target region. For the 2nd PCR, the sense primer was set corresponding to the sequence that is recognised by gRNA and cleaved by Cas9 immediately upstream of the PAM of the target gene, whereas the reverse primer was set on a downstream sequence distant from the PAM. PCR performed using this primer set and the 1 st PCR products as template under standard conditions should generate PCR products (~200 bp) derived from both indel-mutated alleles and unedited alleles. Contrastingly, when the concentration of MgCl 2 used for PCR is lowered, annealing between a primer and the mutated target sequence becomes unstable, and under this condition, PCR products derived from unedited alleles, such as WT allele, should be generated preferentially. Mg 2+ is recognised as not only a cofactor for DNA polymerase activity, but also a promoter of complex formation between primers and DNA template . Blue boxes, sequences recognised by forward and reverse primers used for 1st PCR; green box, sequence recognised by crRNA; red box, PAM sequence. ( b ) Flowchart of detection of biallelic KO mutants by using Bindel-PCR. ( c ) The evaluation rule for indels appearing in the target sequence is shown using mouse Et1 gRNA-recognizing nt region as an example. Green nt correspond to the sequence recognised by gRNA- Et1 . Red nt correspond to the PAM sequence. nt −1 to –23 correspond to the 5′ upstream region of the PAM. nt +1 to +15 correspond to the 3′ downstream region of the PAM. Arrows indicate the site of indels classified. WT, wild-type. ( d ) Analyses of sites frequently showing CRISPR/Cas9-induced indels. Data shown are from our previous study (Sakurai et al ., unpublished data) on mouse Et1 and from other studies [Yoshimi et al . (2016) for rat Tyr, Rosa26, Sirpa , and Cyp2d and Hashimoto and Takemoto (2015) for mouse Fgf10 ]. *Includes the samples with indels at nt (−1) as well as those with indels including a part of the PAM. Et1 , endothelin-1; Tyr , tyrosinase; Rosa26 , Gt(ROSA)26S; Sirpa , signal regulatory protein-α; Cyp2d , cytochrome P450, family 2, subfamily d; Fgf10 , fibroblast growth factor-10.

Article Snippet: For the establishment of Bindel-PCR and for its fidelity test, we used a cloned Taq ( rTaq ) DNA polymerase derived from T. aquaticus DNA polymerase (TaKaRa Taq TM #R001A, TaKaRa) and HiDi DNA polymerase (myPOLS Biotec #9001, myPOLS Biotec GmbH, Konstanz, Germany).

Techniques: CRISPR, Amplification, Sequencing, Derivative Assay, Concentration Assay, Generated, Activity Assay

Journal: Scientific Reports

Article Title: Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

doi: 10.1038/s41598-019-46357-8

Figure Lengend Snippet: Bindel-PCR to identify biallelic KO mutants for Et1 . ( a ) nt sequence in mouse Et1 locus. The location of sequences recognised by gRNA and PCR primers is shown. nt in blue correspond to forward and reverse primers used for 1 st PCR. Green nt correspond to the sequence recognised by gRNA- Et1 . Red nt correspond to the PAM. Expected sizes of PCR products obtained using different primer sets are shown at the bottom. ( b ) nt sequences in mouse Et1 locus that are recognised by gRNA- Et1 . Various types of indels found in a series of Et1 plasmids are shown. nt in green correspond to the sequence recognised by gRNA- Et1 . nt in red correspond to the PAM sequence. Shading indicates modification of Nt. Dashed lines indicate Nt deletion. ( c ) Gel electrophoretic pattern of PCR products (450 bp) obtained through 1 st PCR performed using Et1ch-2S/−2A primer set and a series of Et1 plasmids harbouring various types of indels as template. M, 100-bp-ladder markers. WT, wild-type. The images of these full-length gels are presented in Supplementary Fig. . ( d ) Gel electrophoretic pattern of products from the 2 nd PCR (corresponding to Et1 sequence recognised by gRNA- Et1 ) obtained after performing PCR on a series of Et1 plasmids [shown in ( b ) and ( c )] under reduced concentrations of MgCl 2 . Representative results from assays performed using rTaq DNA polymerase are shown in ( d ). Left panel, 2 nd PCR products (155 bp) obtained using Bindel-PCR(Et1)-1S/-1A primer set; right panel, 2 nd PCR products (153 bp) obtained using Bindel-PCR(Et1)-2S/-1A primer set. Representative results from assays performed using HiDi DNA polymerase are shown in . Left panel, products from the 2 nd PCR (155 bp) obtained using Bindel-PCR(Et1)-1S/-1A primer set; right panel, 2 nd PCR products (153 bp) obtained using Bindel-PCR(Et1)-2S/-1A primer set. M, 100-bp-ladder markers; WT, wild-type. The images of these full-length gels are presented in Supplementary Fig. , .

Article Snippet: For the establishment of Bindel-PCR and for its fidelity test, we used a cloned Taq ( rTaq ) DNA polymerase derived from T. aquaticus DNA polymerase (TaKaRa Taq TM #R001A, TaKaRa) and HiDi DNA polymerase (myPOLS Biotec #9001, myPOLS Biotec GmbH, Konstanz, Germany).

Techniques: Sequencing, Modification

Journal: Scientific Reports

Article Title: Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

doi: 10.1038/s41598-019-46357-8

Figure Lengend Snippet: Analysis of sensitivity for detecting the presence of WT alleles in samples containing various ratios of mutant-type templates. ( a ) PCR of the 1 st PCR templates (shown in Fig. ) mixed at appropriate ratios. For example, Indel Mix 3 was prepared at the ratio of D3-5:I1-1 = 1:1. For preparing WT:Indel M3, WT DNA was added to Indel Mix 3 (D3-5:I1-1 = 1:1) at ratios of 1:1, 1:3, 1:7, 1:15, 1:31, 1:63, and 1:127. Upper panel: Results of 2 nd PCR performed using rTaq DNA polymerase and Bindel-PCR-2S/1A primer set in the presence of 0.85 mM MgCl 2 . Lower panel: Results of 2 nd PCR performed using HiDi DNA polymerase and Bindel-PCR(Et1)-2S/-1A primer set in the presence of 2 mM MgCl 2 . Each gel lane was loaded with 10 μL of PCR products. M, 100-bp-ladder markers. ( b ) WT:Indel samples prepared based on assuming that early mouse embryos are edited by CRISPR/Cas9-related components at these stages: zygote (FE, fertilised egg), 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (M; comprising 16 cells), early blastocyst (B; 32 cells), and blastocyst (64–128 cells). The images of these full-length gels are presented in Supplementary Fig. .

Article Snippet: For the establishment of Bindel-PCR and for its fidelity test, we used a cloned Taq ( rTaq ) DNA polymerase derived from T. aquaticus DNA polymerase (TaKaRa Taq TM #R001A, TaKaRa) and HiDi DNA polymerase (myPOLS Biotec #9001, myPOLS Biotec GmbH, Konstanz, Germany).

Techniques: Mutagenesis, CRISPR

Journal: Scientific Reports

Article Title: Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

doi: 10.1038/s41598-019-46357-8

Figure Lengend Snippet: Identification of biallelic KO mutants among F0 offspring obtained after CRISPR/Cas9-mediated introduction of indels at Et1 locus. ( a ) Sequencing results of genomic DNA samples (10) obtained from the genome-edited F0 offspring that had already been demonstrated to be biallelic KO for Et1 locus (Sakurai et al. , unpublished data). Green nt correspond to the sequence recognised by gRNA- Et1 . Red nt correspond to the PAM sequence. Dashed lines indicate nt deletion. Frequency = no. clones detected/total no. clones sequenced. ( b ) Result of 1 st PCR of genomic DNA samples 1–10, performed using Et1ch-2S/-2A primer set. WT, wild-type genomic DNA used as a positive control; M, 100-bp-ladder markers. Upper panel: Results of 2 nd PCR performed using rTaq DNA polymerase and Bindel-PCR(Et1)-2S/-1A primer set in the presence of 0.85 mM MgCl 2 ; lower panel: results of 2 nd PCR performed using HiDi DNA polymerase and Bindel-PCR(Et1)-2S/-1A primer set in the presence of 2 mM MgCl 2 . WT, wild-type genomic DNA used as a positive control; M, 100-bp-ladder markers. The images of these full-length gels are presented in Supplementary Fig. , .

Article Snippet: For the establishment of Bindel-PCR and for its fidelity test, we used a cloned Taq ( rTaq ) DNA polymerase derived from T. aquaticus DNA polymerase (TaKaRa Taq TM #R001A, TaKaRa) and HiDi DNA polymerase (myPOLS Biotec #9001, myPOLS Biotec GmbH, Konstanz, Germany).

Techniques: CRISPR, Sequencing, Clone Assay, Positive Control

Journal: Scientific Reports

Article Title: Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

doi: 10.1038/s41598-019-46357-8

Figure Lengend Snippet: Identification of biallelic KO mutants among F0 offspring carrying CRISPR/Cas9-induced indels at Tyr ( a ) and Ramp1 loci. ( a ) Location of Tyr sequences recognised by gRNAs and the PCR primers used. Blue, nt sites used as forward and reverse primers for the 1st PCR; green, nt corresponding to gRNA target sequence; red, PAM sequence. Expected sizes of PCR products obtained using different primer sets are shown at the bottom. In the upper right panel, the results of the 1 st PCR of genomic DNA samples of three F0 mice (lanes 1–3; Supplementary Table ) and one F1 offspring (lane 4) are shown when PCR was performed using rTaq DNA polymerase and Tyrch-1S/-1A primer set in the presence of 1.5 mM MgCl 2 . In the lower right panel, the results of the 2 nd PCR using 1 st PCR products are shown when PCR was performed using rTaq DNA polymerase and Bindel-PCR(Tyr)-1S/-1A primer set in the presence of 0.85 mM MgCl 2 . Location of Ramp1 sequences recognised by gRNAs and the PCR primers used. Sites indicated by blue, green, and red correspond to the sites recognised by primers, gRNA, and PAM, respectively. In the upper right panel, the results of the 1 st PCR of genomic DNA samples of eight F0 mice (lanes 1–8; Supplementary Table ) are shown when PCR was performed using rTaq DNA polymerase and Ramp1ch-1S/-1A primer set in the presence of 1.5 mM MgCl 2 . In the lower right panel, the results of the 2 nd PCR of 1 st PCR products are shown when PCR was performed using rTaq DNA polymerase and Bindel-PCR(Ramp1)-1S/-1A primer set in the presence of 0.85 mM MgCl 2 . WT, wild-type genomic DNA used as a positive control; M, 100-bp-ladder markers. Images of these full-length gels are presented in Supplementary Fig. , .

Article Snippet: For the establishment of Bindel-PCR and for its fidelity test, we used a cloned Taq ( rTaq ) DNA polymerase derived from T. aquaticus DNA polymerase (TaKaRa Taq TM #R001A, TaKaRa) and HiDi DNA polymerase (myPOLS Biotec #9001, myPOLS Biotec GmbH, Konstanz, Germany).

Techniques: CRISPR, Sequencing, Positive Control