Journal: The Journal of Physiology

Article Title: Vasopressin casts light on the suprachiasmatic nucleus

doi: 10.1113/JP274025

Figure Lengend Snippet: Secondary and visualization reagents used for retina and SCN

Article Snippet: Supplier Dilution Host Secondary antibody Biotin–anti‐rabbit IgG BA‐1100 Vector Laboratories Ltd, UK 1:500 Horse Biotin–anti‐mouse IgG BA‐2001 Vector Laboratories Ltd, UK 1:500 Horse Biotin–anti‐guinea pig IgG BA‐7000 Vector Laboratories Ltd, UK 1:500 Goat Biotin–anti‐rabbit IgG BA‐1000 Vector Laboratories Ltd, UK 1:500 Goat Biotin–anti‐mouse IgG BA‐9200 Vector Laboratories Ltd, UK 1:500 Goat Alexa 488 anti‐chicken A11039 Life Technologies, UK 1:500 Goat Biotin–anti‐chicken IgY 703‐066‐155 Stratech Scientific Ltd, UK 1:500 Donkey Visualized with Streptavidin, Alexa Fluor 488 conjugate S‐11223 Life Technologies, UK 1:500 Streptavidin, Alexa Fluor 555 conjugate S‐21381 Life Technologies, UK 1:500 Streptavidin, Alexa Fluor 647 conjugate S‐21374 Life Technologies, UK 1:500 Open in a separate window Secondary and visualization reagents used for retina and SCN Microscopy Fluorescence signals were acquired either using a Nikon AIR confocal or a Zeiss LSM510 Axiovert confocal laser scanning microscope.

Techniques: Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates the Wnt/β-catenin pathway by stabilizing β-catenin. A-B , MYC, CCND1 and nuclear β-catenin levels in NSCLC cells as indicated were analyzed by western blotting. C , CTNNB1 transcript levels in NSCLC cells as indicated was analyzed by qRT-PCR. ns, not significant. D , Subcellular localization of SETD1A and β-catenin in NSCLC cells was analyzed by confocal laser scanning microscope. The subcellular distribution of SETD1A and β-catenin was quantified by Image J software. E , The interaction between SETD1A and β-catenin in PC9 cells was analyzed by CoIP assay. F - G , β-catenin stability in the SETD1A knockdown and negative control group PC9 cells was analyzed by CHX chase assay. H , Ubiquitination of β-catenin in PC9 cells was analyzed by CoIP assay following SETD1A knockdown. I , Two-step co-immunoprecipitation of the complex containing SETD1A, β-catenin and PKA is shown. HEK293T cells were cotransfected with the Flag-tagged SETD1A plasmid and HA-tagged β-catenin plasmid. The first immunoprecipitation was performed using anti-FLAG M2 beads. The complex was eluted using 3 × Flag peptide followed by the second step of co-immunoprecipitation with anti-HA antibody. Then the protein samples were analyzed by western blotting with anti-Flag, anti-HA and anti- anti-PKAα cat antibody. Data are shown as means ± SD.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Western Blot, Quantitative RT-PCR, Laser-Scanning Microscopy, Software, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A binds to β-catenin via its SET domain, A , Schematic representation of SETD1A mutants. B , HEK293T cells were cotransfected with the indicated plasmids of Flag-tagged SETD1A mutants and HA-tagged full length β-catenin. Cell lysates were immunoprecipitated with anti-Flag antibody. C , The total, cytoplasmic and nuclear β-catenin levels were detected by western blot analysis following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. D , Sphere formation ability was determined following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. Scale bar, 100 μm. E , Cisplatin sensitivity was detected by the CCK-8 assay following transfection with the empty vector, wild type SETD1A plasmid and ΔSET plasmid. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates NEAT1 and EZH2 transcription to activate the Wnt/β-catenin pathway. A-B , The correlation between SETD1A and NEAT1, EZH2 was analyzed using GEPIA online tool. R, Pearson’s correlation coefficient. C , Schematic showing the ChIP-PCR detection site in the NEAT1 and EZH2 promoters. D , The enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters in A549 cells was detected by ChIP-PCR assay. E , The relative enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters was detected by ChIP-qPCR assay following SETD1A knockdown in A549 cells. F , The promoter activity was analyzed by luciferase activity assay in A549 cells. G - H , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A knockdown. I , EZH2 protein levels in NSCLC cells as indicated were detected by western blotting following SETD1A knockdown. J-K , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A overexpression. L , EZH2 protein levels in NSCLC cells were detected by western blotting following SETD1A overexpression. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: The SETD1A/β-catenin axis promotes NSCLC progression in vivo. A , A photograph of the tumors collected from nude mice after 28 days ( n = 5). B , The tumor volumes were measured at the indicated time points. C , The tumor weights were measured after xenograft resection. D , Immunohistochemical staining of Ki67, EZH2 and β-catenin in the xenograft specimens. Scale bar, 50 μm. E , The transcript levels of NEAT1, EZH2 and CTNNB1 in the xenograft tissues were detected by qRT-PCR. F , Tumor initiation was monitored for 8 weeks and the tumor initiation frequency was calculated using the ELDA. Data are shown as means ± SD. ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: In Vivo, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A is a direct target of the Wnt/β-catenin pathway. A , Schematic showing the putative TCF/LEF binding site in the TCF7L2/TCF4 enriched region. Red line, putative TCF/LEF binding site predicted by PROMO online tool; green region, TCF7L2/TCF4 enriched region derived from ENCODE database. B , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-PCR assay. C , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-qPCR assay. D , The promoter activity of SETD1A gene in NSCLC cells was analyzed by luciferase activity assay. ns, not significant. E , SETD1A transcript levels in NSCLC cell as indicated were detected by qRT-PCR. H, SETD1A protein levels in NSCLC cells as indicated were detected by western blotting. Data are shown as the means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Binding Assay, Derivative Assay, Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: Schematic diagram of the SETD1A/Wnt/β-catenin positive feedback loop in this study. In NSCLC cells, SETD1A activates the Wnt/β-catenin pathway through two mechanisms as follows: i, SETD1A interacts with and stabilizes β-catenin protein; ii, SETD1A promotes the transcription of NEAT1 and EZH2. In turn, the Wnt/β-catenin pathway activates SETD1A transcription, thus forming a positive feedback loop to coordinately promote NSCLC progression

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques:

Journal: STAR Protocols

Article Title: Protocol for inducing cellular ablation in the mouse atrioventricular conduction system

doi: 10.1016/j.xpro.2023.102145

Figure Lengend Snippet: Immunohistochemical staining of the AV node (A) X-Gal staining of AVCS-iLacZ (Tg Cx30.2-MerCreMer/+ ; Rosa26 LacZ/+ ) mouse heart, induced at P0 and harvested at P28, scale bar: 500 μm. (B) X-Gal staining of the AV node of the AVCS-iLacZ mouse, scale bar: 200 μm. (C) Acetylcholine esterase staining of the AV node of the AVCS-iLacZ, scale bar: 200 μm. (D) Immunofluorescence staining of the AV node of the AVCS-iLacZ mouse using HCN4 antibody, scale bar: 200 μm.

Article Snippet: Polyclonal anti-HCN4 guinea pig antibody (1:200 dilution) , Alomone Labs , Cat#AGP-004.

Techniques: Immunohistochemical staining, Staining, Immunofluorescence

Journal: STAR Protocols

Article Title: Protocol for inducing cellular ablation in the mouse atrioventricular conduction system

doi: 10.1016/j.xpro.2023.102145

Figure Lengend Snippet: Cellular ablation of the AV node of iAVB (Tg Cx30.2-MerCreMer/+ ; Rosa26 DTA/LacZ ) mouse heart after Tamoxifen induction (A and B) Masson’s trichrome staining of the AV node of iAVB mouse compared to control, scale bar: 100 μm. (C) Quantification of the AVN fibrosis of iAVB mouse compared to control, ∗p-value <0.05. Error bars represent SEM. (D and E) HCN4 immunofluorescence staining of the AV node of iAVB mouse compared to control, scale bar: 100 μm. (F) Quantification of HCN4 positive cell dropout in the AVN of iAVB mouse compared to control, ∗∗p-value <0.01. Error bars represent SEM.

Article Snippet: Polyclonal anti-HCN4 guinea pig antibody (1:200 dilution) , Alomone Labs , Cat#AGP-004.

Techniques: Staining, Control, Immunofluorescence

Journal: STAR Protocols

Article Title: Protocol for inducing cellular ablation in the mouse atrioventricular conduction system

doi: 10.1016/j.xpro.2023.102145

Figure Lengend Snippet:

Article Snippet: Polyclonal anti-HCN4 guinea pig antibody (1:200 dilution) , Alomone Labs , Cat#AGP-004.

Techniques: Recombinant, Electron Microscopy, Blocking Assay, Saline, Plasmid Preparation, Staining, Modification, Software, Laser-Scanning Microscopy

Journal: STAR Protocols

Article Title: Protocol for inducing cellular ablation in the mouse atrioventricular conduction system

doi: 10.1016/j.xpro.2023.102145

Figure Lengend Snippet: Preparation of primary antibody

Article Snippet: Polyclonal anti-HCN4 guinea pig antibody (1:200 dilution) , Alomone Labs , Cat#AGP-004.

Techniques: Concentration Assay, Blocking Assay

Journal: Virus genes

Article Title: Subcellular localization of the porcine deltacoronavirus nucleocapsid protein.

doi: 10.1007/s11262-020-01790-0

Figure Lengend Snippet: Fig. 2 PDCoV N protein distributes in both the nucleolus and cytoplasm. a LLC-PK1 cells transfected with pCAGGS- HA-PDCoV-N plasmids were subjected to western blotting using anti-HA antibodies or β-actin antibodies. b LLC- PK1 cells were transfected with pCAGGS-HA-PDCoV- N vectors or infected with PDCoV, then fixed with Triton X-100. After incubation with mouse monoclonal antibod- ies against HA or PDCoV-N protein and rabbit polyclonal antibody against B23, and then cells were stained with DAPI. The cells were observed and photographed using a confocal laser scanning microscope. The N protein is colored green, and B23 is colored red

Article Snippet: Rabbit anti-B23 antibody was purchased from Proteintech (CHI, USA).

Techniques: Transfection, Western Blot, Infection, Incubation, Staining, Laser-Scanning Microscopy

Journal: Scientific Reports

Article Title: Loss of Dead end1 induces testicular teratomas from primordial germ cells that failed to undergo sexual differentiation in embryonic testes

doi: 10.1038/s41598-023-33706-x

Figure Lengend Snippet: Dnd1 deletion in migrating PGCs causes testicular teratomas. ( A ), Mating strategy for the generation of conditional knockout mice of Dnd1 . ( B ), Comparison of testis size between 4-week-old littermates of control ( Dnd1 flox/flox ) and Dnd1 -cKO ( Dnd1 flox/flox ; Oct4ΔPE-CreER T2 ) mice. ( C – G) , Testis sections from 4-week-old control ( C ) and Dnd1 -cKO ( D – G ) mice were stained with haematoxylin and eosin. Hair follicles, muscles, and gut-like epithelium are observed in ( E , F ), and ( G ), respectively. ( H , I ), Testis from control ( H ) and Dnd1 -cKO ( I ) embryos with Nanog-GFP transgene at E16.5, under a fluorescence microscope. The arrowheads in ( I ) indicate GFP-positive foci. ( J – R ), Testis sections from E16.5 Dnd1 -cKO embryos with Nanog-GFP transgene were immunostained with antibodies against GFP ( J , M , P ), NANOG ( K ), POU5F1 ( N ), and OTX2 ( Q ). DNA was labelled with DAPI (blue) ( L , O , R ). Images correspond to one of n = 3 biological replicates. S , The incidence of male embryos with at least one ECC colony in the testes was analysed in control and Dnd1 -cKO embryos with Nanog-GFP transgene after injection of tamoxifen at E10.5, E11.5, and E12.5. Scale bars: 5 mm in ( B ), 100 μm in ( C ) and ( D ), 100 μm in ( E – G ), 200 μm in ( H ) and ( I ), and 100 μm in ( J – R ).

Article Snippet: After deparaffinisation, the sections were autoclaved for 15 min at 105 °C with Antigen Unmasking Solution (H-3300, Vector Laboratories, CA) followed by blocking with 5% skim milk for 30 min, and then incubated overnight at 4 °C with primary antibodies against DAZL (1:2,000) produced by a guinea pig , DND1 (1: 2,000) produced by a guinea pig , NANOS2 (1:200) produced by a rabbit , GFP (1: 1000; NB100-1770, Novus Biological, CO), NANOG (1:1000; IHC-00205, BETHYL Laboratories, MA), OCT4/POU5F1 (1:2000; sc-5279, Santa Cruz, TX), OTX2 (1:2,400; EPR20375, Abcam, UK), KI67 (1:2,000; RM-9106-S0, Thermo Fisher Scientific, MA), LAMININ (1:1,000; LAM-1, ICN Biomedicals, PA), cleaved CASPASE3 (1: 900; Asp175, Cell Signaling Technology, MA), or SYCP3 (1: 400; a gift from Dr. Chuma) .

Techniques: Knock-Out, Staining, Fluorescence, Microscopy, Injection

Journal: Journal of Virology

Article Title: Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

doi: 10.1128/JVI.01350-20

Figure Lengend Snippet: PRRSV particles are transported along the endosome-lysosome pathway during an early stage of infection. Marc-145 cells or PAMs were prechilled on ice for 30 min before incubation with 1.0 MOI of PRRSV. Cells were further incubated on ice for 1 h to ensure abundant adsorption of virus. Subsequently, cells were washed extensively with ice-cold PBS to remove unabsorbed virus and incubated at 37°C for the indicated time points. Cells were fixed with 70% −20°C prechilled alcohol, and cellular compartments and viral proteins were stained with specific antibodies against early endosomes (Rab5), late endosomes (M6PR), lysosomes (LAMP2), and capsid protein (N protein), followed by staining with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) antibody to visualize cellular compartments (green) and Alexa Fluor-594 conjugated goat anti-mouse IgG (H&L) antibody to visualize virions (red). Fluorescent images were acquired with a confocal laser scanning microscope. Scale bar, 10 μm. (A and C) Confocal immunofluorescence images showing PRRSV particles colocalized with endosomes and lysosomes at different time points. (B and D) Colocalization analysis corresponding to the PRRSV particles and cellular compartments. PAM, porcine alveolar macrophage; PRRSV, porcine reproductive and respiratory syndrome virus; MOI, multiplicity of infection.

Article Snippet: Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) and Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) were obtained from Abcam.

Techniques: Infection, Incubation, Adsorption, Staining, Laser-Scanning Microscopy, Immunofluorescence

Journal: Journal of Virology

Article Title: Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

doi: 10.1128/JVI.01350-20

Figure Lengend Snippet: IFITM3 does not affect PRRSV particle attachment, entry, or access to endosomes or lysosomes. (A) Marc-145-Vector or Marc-145-IFITM3-flag cells were incubated with PRRSV at an MOI of 1.0 for 6, 8, 10, and 12 h. The infected cells were fixed and stained with anti-PRRSV N antibody and counterstained with DAPI to visualize the nuclei. (B) Marc-145-Vector or Marc-145-IFITM3-flag cells were infected with 1.0 MOI of PRRSV for 1 h at 37°C, and then cells were washed with PBS and cultured with 3% FBS+DMEM. Cells were harvested 1, 2, 4, 5, 6, 8, or 10 hpi, and PRRSV genome levels were determined. Prechilled Marc-145-Vector or Marc-145-IFITM3-flag cells were infected with PRRSV (1.0 MOI) and further chilled on ice for 1 h. (C) For the attachment assay, after washing 3 times using ice-cold PBS, cells were harvested for PRRSV genome abundance analysis. (D) For the entry assay, cells were washed with PBS three times and treated with 37°C prewarmed DMEM and incubated at 37°C for 1 h, followed by trypsin treatment for 30 sec and 3 washes with PBS. Cells were collected for PRRSV genome content analysis. (E) Marc-145-Vector or Marc-145-IFITM3-flag cells were inoculated with 1.0 MOI PRRSV for 1 h at 37°C, and then cells were fixed and stained with anti-PRRSV N, -Rab5, -M6PR, and -LAMP2 antibodies, followed by an Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) antibody and an Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) antibody. Colocalization of PRRSV particles with cellular vesicles was quantified, and representative images are presented. Scale bar, 10 μm. Horizontal bars represent the mean of the Pearson’s correlation coefficient (Rr) calculated based on 10 fields of view, with error bars marking the 95% confidence intervals. PRRSV, porcine reproductive and respiratory syndrome virus; MOI, multiplicity of infection; hpi, hours postinfection.

Article Snippet: Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) and Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) were obtained from Abcam.

Techniques: Plasmid Preparation, Incubation, Infection, Staining, Cell Culture

Journal: Journal of Virology

Article Title: Interferon-Induced Transmembrane Protein 3 Is a Virus-Associated Protein Which Suppresses Porcine Reproductive and Respiratory Syndrome Virus Replication by Blocking Viral Membrane Fusion

doi: 10.1128/JVI.01350-20

Figure Lengend Snippet: PRRSV particles are transported to IFITM3-containing endosomes or lysosomes. (A) Marc-145-IFITM3-flag cells transfected with pcDNA3.1-Rab5-GFP, -Rab7, and -LAMP1-YFP plasmids for 48 h were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were labeled with anti-IFITM3 and -Rab7 antibodies followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) antibody for IFITM3 (red) and Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) antibody for Rab7 (green), and cell nuclei were counterstained using DAPI. Rab5 and LAMP1 (green) were observed directly using a confocal microscope. (B) PRRSV were allowed to bind to prechilled Marc-145-IFITM3-flag cells for 1 h on ice, unbound virions were washed, and cells were warmed to 37°C for 45 min. IFITM3 was stained using an anti-IFITM3 antibody and the corresponding fluorescent second antibody (blue), and PRRSV was stained using an anti-N antibody and a fluorescent second antibody (red). (C) Marc-145-IFITM3-flag cells were infected with 0.1 MOI of PRRSV for 12 h, and then cells were transfected with pcDNA3.1-Rab5-GFP, -Rab7, and -LAMP1-YFP plasmids for 36 h and fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Cells were labeled as described in . Scale bar, 10 μM. PRRSV, porcine reproductive and respiratory syndrome virus; IFITM, interferon-induced transmembrane.

Article Snippet: Alexa Fluor 488-conjugated goat anti-rabbit IgG (H&L) and Alexa Fluor 594-conjugated goat anti-mouse IgG (H&L) were obtained from Abcam.

Techniques: Transfection, Labeling, Microscopy, Staining, Infection