Journal: STAR Protocols

Article Title: Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy

doi: 10.1016/j.xpro.2023.102552

Figure Lengend Snippet: Cross-sectional diagram of sample mount This protocol for heart muscle cells uses an inverted microscope with a Nikon 100× Plan Apo, Lambda, oil 0.13 mm working distance lens.

Article Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Techniques: Inverted Microscopy

Journal: STAR Protocols

Article Title: Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy

doi: 10.1016/j.xpro.2023.102552

Figure Lengend Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Article Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Techniques: Microscopy

Journal: STAR Protocols

Article Title: Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy

doi: 10.1016/j.xpro.2023.102552

Figure Lengend Snippet: Acquiring Z-stack image Next step of appropriately saturated nucleus in Figure 4 B (A) Screen shot of the ND Acquisition window to demonstrate setting z-stack parameters. (B) Z-stack ND file image of neonatal rat cardiomyocyte acquired using Plan Apo Lambda 100× oil 1.45NA, 0.133 mm lens objective. Scale bar 2 μm. (C) 2D Max projection image of scanned nucleus showing merged image along with separate channels. Scale bar 2 μm. (D) Look Up Table (LUT) indicating appropriate saturation levels.

Article Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Techniques:

Journal: STAR Protocols

Article Title: Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy

doi: 10.1016/j.xpro.2023.102552

Figure Lengend Snippet: High resolution image of nuclear pores stained with Mab NUP 153 Induced pluripotent stem cell cardiomyocytes (ips-cm’s) acquired using Plan Apo Lambda 100× oil 1.45NA, 0.13 mm objective lens. Red arrows indicate high nuclear pore antibody signal. Yellow arrows indicate low nuclear pore antibody signal. Scale bar 10 μm.

Article Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Techniques: Staining

Journal: STAR Protocols

Article Title: Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy

doi: 10.1016/j.xpro.2023.102552

Figure Lengend Snippet: Zoomed in high resolution image of nuclear pores stained with Mab NUP 153 Mouse cardiomyocyte nucleus acquired using Plan Apo Lambda 100× oil 1.45NA, 0.13 mm objective lens showing high background staining. Scale bar 5 μm.

Article Snippet: Neonatal rat cardiomyocyte imaged on high resolution with a Plan Apo Lambda 100× oil lens objective using Nikon A1R scanning laser confocal microscope Scale bar 10 μm.

Techniques: Staining

Journal: PLoS Pathogens

Article Title: EBV Latent Membrane Protein 1 Activates Akt, NFκB, and Stat3 in B Cell Lymphomas

doi: 10.1371/journal.ppat.0030166

Figure Lengend Snippet: (A) Relative expression of IL10, IL15, and IFNγ mRNA in WT and LMP1 transgenic B cells (CD19+), as detected with an Rnase protection assay. Mouse lymphoma cell lines 967 and K46μ were used as controls. Expression levels were quantified with a phosphorimager and values were normalized to the ribosomal housekeeping gene L32. The cytokine:L32 ratio was set to 1 in the mouse B cell lymphoma line 967. (B and C) Immunoblot analysis of activated pStat3 in purified B cells (CD19+) from WT and LMP1 transgenic mice (B) at the time of harvest, and (C) 4 h after culture with or without IL10, a neutralizing antibody to IL10, or a rat IgG1 isotype control. (C) Shown are the results for WT lymphoma 1 and LMP1 transgenic lymphoma 1. Arrows indicate the positions of the α and β isoforms of Stat3. Actin was used as a loading control. (D) Immunohistochemistry detection of activated nuclear pStat3 in the spleens of WT and LMP1 transgenic mice. Scale bar, 20 μm.

Article Snippet: FITC-conjugated goat anti-mouse IgM, rat IgG2aκ anti-mouse IgD (clone 11–26), rat IgG1κ anti-mouse kappa (clone 187.1), rat IgG2bκ anti-mouse lambda (clone JC5–1); PE-conjugated goat anti-mouse IgG; un-conjugated goat anti-mouse kappa and anti-mouse lambda were purchased from Southern Biotech.

Techniques: Expressing, Transgenic Assay, Rnase Protection Assay, Western Blot, Purification, Control, Immunohistochemistry

Journal: Cells

Article Title: Nuclear Progestin Receptor Phosphorylation by Cdk9 Is Required for the Expression of Mmp15, a Protease Indispensable for Ovulation in Medaka

doi: 10.3390/cells8030215

Figure Lengend Snippet: Roscovitine inhibition of Pgr phosphorylation in preovulatory follicles. ( A ) The −14 h-follicles were incubated with roscovitine (Rosc) at 0.5, 5, or 50 μM for 14 h, and the follicle layer extracts were analyzed by western blotting. ( B ) Preovulatory follicles were isolated at various time points, and their extracts were analyzed by immunoprecipitation/Phos-tag SDS-PAGE/western blotting (upper panel) and immunoprecipitation/regular SDS-PAGE/western blotting using an antibody for medaka Pgr (lower panel). Phosphorylated (indicated by arrow) and unphosphorylated forms of Pgr (indicated by arrowhead) are shown in the upper panel. An asterisk indicates the bands corresponding to the antibody used for immunoprecipitation. As controls, the −7 h-follicle extracts were immunoprecipitated using normal IgG. ( C ) The materials immunoprecipitated from follicle extracts obtained from the −7 h-follicles were incubated with or without phosphatase, and the samples were analyzed by Phos-tag SDS-PAGE/western blotting using the antibody for medaka Pgr. Phosphorylated (indicated by arrow) and unphosphorylated forms of Pgr (indicated by arrowhead) are shown. An asterisk indicates the bands corresponding to the antibody used for immunoprecipitation. ( D ) The −14 h-follicles were incubated for 14 h with Rosc (50 μM), with MEK inhibitor (10 μM), or without any additives, and the follicle extracts of the treated follicles were analyzed by immunoprecipitation/Phos-tag SDS-PAGE/western blotting using the antibody for medaka Pgr. Phosphorylated (indicate by arrow) and unphosphorylated forms of Pgr (indicated by arrowhead) are shown. An asterisk indicates the bands corresponding to the antibody used for immunoprecipitation.

Article Snippet: The materials immunoprecipitated using rat anti-medaka Pgr antibody were incubated with or without Lambda Protein Phosphatase (New England BioLabs Inc., Ipswich, MA, USA) for 60 min at 30 °C in 1 × NEB Buffer for Protein Metallo Phosphatase supplemented with 1 mM MnCl 2 and gentle agitation.

Techniques: Inhibition, Incubation, Western Blot, Isolation, Immunoprecipitation, SDS Page

Journal: Cells

Article Title: Nuclear Progestin Receptor Phosphorylation by Cdk9 Is Required for the Expression of Mmp15, a Protease Indispensable for Ovulation in Medaka

doi: 10.3390/cells8030215

Figure Lengend Snippet: Effect of roscovitine treatment of follicles on Pgr binding to the mmp15 promoter region. ( A ) For ChIP assays to examine the binding of Pgr to mmp15 promoter region, seven ChIP primer pairs (P1–P7) in the 1.5 kb upstream region of the transcription start site (indicated as +1) of the mmp15 gene and another primer pair (P8 as a negative control) were prepared. Putative progestin receptor elements (PREs) in the region are indicated by boxes. ( B ) The −14 h-follicles were incubated in vitro without roscovitine (Rosc) for 14 h, and the resulting follicles were used for ChIP assays and amplified with primer pairs P1 to P8. The sheared DNA immunoprecipitated with anti-medaka Pgr antibody was analyzed by real-time RT-PCR. Asterisks indicate a significant difference at p < 0.01 (**) compared to the negative control (ANOVA and Dunnett’s post hoc test, N = 4). ( C ) The −14 h-follicles were incubated in vitro for 7 h or 14 h, and the resulting follicles were used for ChIP assays using primer pair P1. The sheared DNA immunoprecipitated with anti-medaka Pgr antibody was analyzed by real-time RT-PCR. Asterisks indicate a significant difference at p < 0.01 (**) compared with the 7 h-incubated follicles ( t -test, N = 4). ( D ) The −14 h-follicles were incubated in vitro with Rosc (50 μM) for 14 h, and the resulting follicles were used in ChIP assays as in ( B ). ( E ) OLHNI-2 cells stably expressing medaka Pgr were cultured alone, in the presence of 17,20βP (100nM), or in the presence of both 17,20βP and Rosc. After culturing for 24 h, the expression levels of mmp15 were examined by real-time RT-PCR. Asterisks indicate a significant difference at p < 0.05 (*) (ANOVA and Dunnett’s post hoc test, N = 4).

Article Snippet: The materials immunoprecipitated using rat anti-medaka Pgr antibody were incubated with or without Lambda Protein Phosphatase (New England BioLabs Inc., Ipswich, MA, USA) for 60 min at 30 °C in 1 × NEB Buffer for Protein Metallo Phosphatase supplemented with 1 mM MnCl 2 and gentle agitation.

Techniques: Binding Assay, Negative Control, Incubation, In Vitro, Amplification, Immunoprecipitation, Quantitative RT-PCR, Stable Transfection, Expressing, Cell Culture

Journal: Cells

Article Title: Nuclear Progestin Receptor Phosphorylation by Cdk9 Is Required for the Expression of Mmp15, a Protease Indispensable for Ovulation in Medaka

doi: 10.3390/cells8030215

Figure Lengend Snippet: Further evidence for the role of Cdk9/Ccni in the follicles that are destined to ovulate. ( A ) The −14 h-follicles were incubated in vitro with the specific CDK9 inhibitor, CDK9-inhibitor II, at 1 and 10 μM for 18 h, and the rate of ovulation was determined. Asterisks indicate significance at p < 0.01 (**) compared to follicles incubated without any additives (ANOVA and Dunnett’s post hoc test, N = 5). ( B ) Incubation of follicles with CDK9-inhibitor II was conducted as in ( A ) except the duration of incubation was 14 h. Note that the inhibitor had no effect on the GVBD of follicles (N = 5). ( C ) The -14 h-follicles were incubated in vitro with or without the specific CDK9 inhibitor CDK9-inhibitor II (10 μM) for 18 h, and the expression of Mmp15 in the follicle layer of the follicles was analyzed by western blotting. ( D ) The −14 h-follicles were incubated in vitro with or without the specific CDK9 inhibitor CDK9-inhibitor II (10 μM) for 14 h, and Pgr phosphorylation in the follicles was examined by immunoprecipitation/Phos-tag SDS-PAGE/western blot analysis. Positions of phosphorylated (indicated by arrow) and unphosphorylated Pgr (indicated by arrowhead) are shown. As control, extracts of the follicles treated with CDK9-inhibitor II were immunoprecipitated with normal IgG. An asterisk indicates the bands corresponding to the antibody used for immunoprecipitation. ( E ) OLHNI-2 cells stably expressing medaka Pgr (Cont) and Cdk9-deficient OLHNI-2 cells (Cdk9 KO), which were generated from the above cells with CRISPR/Cas9 technology, were immunoprecipitated with medaka Pgr antibody, and the resulting precipitated materials were analyzed by Phos-tag SDS-PAGE/western blot analysis. Positions of phosphorylated (indicated by arrow) and unphosphorylated Pgr (indicated by arrowhead) are shown. As a control, extracts of Cdk9-deficient OLHNI-2 cells were immunoprecipitated with normal IgG. An asterisk indicates the bands corresponding to the antibody used for immunoprecipitation. ( F ) Extracts of the −6 h-follicles were immunoprecipitated with medaka Cdk9 antibody or normal IgG, and the resulting precipitated materials were then analyzed by western blotting using the same Cdk9 antibody (left two lanes) or medaka Ccni antibody (right two lanes). Asterisks indicate the bands corresponding to the antibody used for immunoprecipitation.

Article Snippet: The materials immunoprecipitated using rat anti-medaka Pgr antibody were incubated with or without Lambda Protein Phosphatase (New England BioLabs Inc., Ipswich, MA, USA) for 60 min at 30 °C in 1 × NEB Buffer for Protein Metallo Phosphatase supplemented with 1 mM MnCl 2 and gentle agitation.

Techniques: Incubation, In Vitro, Expressing, Western Blot, Immunoprecipitation, SDS Page, Stable Transfection, Generated, CRISPR