Journal: Scientific reports

Article Title: ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease.

doi: 10.1038/s41598-018-23034-w

Figure Lengend Snippet: Figure 3. Screening the canine cohort for ILF3 and RBMX autoantibodies. A radio-ligand binding assay was used to screen for ILF3 (a) and RBMX (b) autoantibodies in dogs. The cutoff value for ILF3 was calculated from healthy controls as the mean + 5 SD. For RBMX, the mean + 7 SD was used as cutoff value because of a clear separation of negative and positive samples. All samples were analysed in duplicate. Autoantibody index = (sample value mean − negative control)/(positive control − negative control) * 100. NSDTR, Nova Scotia duck tolling retriever. IMRD ANAS, IMRD patients with speckled antinuclear antibodies (ANA) pattern. IMRD ANAH, IMRD patients with homogenous ANA pattern. IMRD ANAneg, IMRD patients without antinuclear antibodies. SRMA, steroid-responsive meningitis-arteritis.

Article Snippet: Human ILF3 cDNA in a pCMV6-Entry vector (RC214999, Origene) was used for in vitro transcription and translation of ILF3.

Techniques: Radio Ligand Binding Assay, Negative Control, Positive Control

Journal: Scientific reports

Article Title: ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease.

doi: 10.1038/s41598-018-23034-w

Figure Lengend Snippet: Figure 5. Sera from ILF2- and ILF3-positive patients and commercial ILF2 and ILF3 antibodies display a speckled ANA pattern. Indirect immunofluorescence microscopy images of HEp-2 cells incubated with sera from an ILF2- and ILF3-positive IMRD patient (a), a commercial polyclonal ILF2 antibody (b); and a commercial polyclonal ILF3 antibody (c). (a) In the nucleoplasm, fine tiny speckles can be observed and mitotic cells have unstained chromatin mass (⇨). The nucleoli are not stained (→). (b,c) In the nucleoplasm, fine tiny speckles can be observed and mitotic cells have unstained chromatin mass (⇨). Nucleoli are stained in many of the cells (→) but not all.

Article Snippet: Human ILF3 cDNA in a pCMV6-Entry vector (RC214999, Origene) was used for in vitro transcription and translation of ILF3.

Techniques: Immunofluorescence, Microscopy, Incubation, Staining

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: BoHV-1 infection has no obvious effects on NFAT5 steady-state protein levels at late stages after infection. (A) Confluent MDBK cells in 60 mm dishes were infected with BoHV-1 at an MOI of 0.1 for 16 h and 24 h (h). Cells were lysed with RIPA buffer and then analyzed by Western blotting to detect NFAT5 protein levels. β-Actin was detected as a loading control. (B) Band intensity was analyzed with free software Image J. Each analysis was compared with that of mock-infected controls at indicated times after infection, which was arbitrarily set as 1. (C) MDBK cells in 6-well plates were either mock-infected or infected with BoHV-1 at an MOI of 0.1. At 24 h postinfection, the total RNA was purified, and the mRNA levels of NFAT5 were examined with qRT-PCR. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with a Student's t test ( *, P < 0.05; ns, not significant).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Western Blot, Control, Software, Purification, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of BoHV-1 infection had on NFAT5 localization. MDBK cells in a 2-well chamber slide were either mock-infected or infected with BoHV-1 at an MOI of 0.1. After infection for 24 h, the cells were fixed with 4% formaldehyde, and NFAT5 protein was immunostained by using NFAT5-specific MAb via IFA. DAPI staining was used to stain nuclear DNA. Images were obtained by performing confocal microscopy (Leica).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Staining, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on the accumulation of NFAT5 in mitochondria. (A) MDBK cells in 100 mm dishes were either mock-infected or infected with BoHV-1 (MOI = 0.1) for 24 h. The cells were collected for isolation of mitochondria fractions via using a commercial kit (Beyotime Biotechnology, cat# C3601) following the manufacturer’s protocol. Protein levels of NFAT5 were detected by Western blotting. HSP60 was detected and used as a protein loading control. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 1. The results shown are representations of three independent experiments. Error bars denote the variability between three independent experiments. Significance was assessed with a Student's t test (*, P < 0.05).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Isolation, Western Blot, Control, Software

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on nuclear accumulation of NFAT5 proteins. (A) MDBK cells in 100 mm dishes were either mock-infected or infected with BoHV-1 (MOI = 0.1) for 24 h. The cells were collected for isolation of both nucleus and cytosol fractions via using a commercial kit (Beyotime Biotechnology, cat# P0027), following the manufacturer’s protocol. Protein levels of NFAT5 were detected by Western blotting. LaminA/C, a marker for nuclear protein, and β-tubulin, a marker for cytoplasmic protein, were detected as a protein loading control, respectively. The results shown are representations of three independent experiments.

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Isolation, Western Blot, Marker, Control

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on the transcription of NFAT5 downstream targets. MDBK cells in 6-well plates were either mock-infected or infected with BoHV-1 at an MOI of 0.1. At 24 h postinfection, total RNA was purified, and the mRNA levels of PGK1 (A), SMIT (B), and BGT-1 (C) were examined with qRT-PCR. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with a Student's t test ( *, P < 0.05).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Purification, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: NFAT5-specific siRNA increases virus production in MDBK cells. (A) MDBK cells in 6-well plates were transfected with either scrambled siRNA (200 pmol) or three individual siRNA targeting NFAT5 (200 pmol), referred to as siRNANFAT5-1, siRNANFAT5-2, and siRNANFAT5-3, respectively. At 48 h after transfection, the effective siRNA were screened by detection of NFAT5 protein levels via Western blot. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 100%. (C and D) MDBK cells in six-well plates were transfected with either scrambled siRNA (200 pmol) or siRNANFAT5-3 (200 pmol). After transfection for 48 h, cell cultures were infected with BoHV-1 (MOI = 0.1). After infection for 24 h, the cellular genome was purified by using a commercial viral DNA purification kit, and the viral genome was examined by using qPCR using gC-specific primer(C). In parallel, cell infection virus yield in supernatants was examined with results expressed as TCID 50 /mL (D). (E and F) NFAT5 plasmid of 1 and 2.5 μg along with empty vector was transfected into MDBK cells in 6-well plates using, after transfection for 48 h, the cells were either collected for the detection of NFAT5 protein levels using Western blot (E), or subjected to virus infection (MOI = 0.1), and the virus production were tittered in MDBK cells with results expressed as TCID 50 /mL (F). The data represented three independent experiments. Results are the mean of three independent experiments, with error bars showing standard deviations. Significance was assessed with a Student's t test (*, P < 0.05; ns, not significant).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Virus, Transfection, Western Blot, Software, Control, Infection, Purification, DNA Purification, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of NFAT5-specific inhibitor KRN5 had on BoHV-1 productive infection. (A) MDBK cells in 24-well plates were treated with either DMSO control or 10 μM KRN5. After treatment for 24 h, the cytotoxicity in MDBK cells was analyzed with a Trypan-blue exclusion test. (B) Confluent MDBK cells in 24-well plates were pretreated with either DMSO control or KRN5 at a concentration ranging from 0.5 to 10 μM at 2 h before infection. The cells were infected with BoHV-1 at MOI of 0.1 for 1 h in the presence of either DMSO or KRN5. After extensive washing with PBS, a fresh medium containing inhibitors or DMSO control was replaced. At 12 and 24 h postinfection, the cell cultures were collected, and virus titers were measured with results expressed as TCID 50 /mL. (C to E) As described in panel B, MDBK were treated with 2 μM KRN5 during virus infection plus a pretreatment for 2 h prior to infection. After infection for 24 h, total RNA was isolated and analyzed by qRT-PCR to measure viral mRNA transcripts of bICP0 (C), DNA polymerase (D), and gC (E) by using relative qRT-PCR. (F) As described in panels C to E, MDBK cells in 6-well plates were either mock infected or infected with BoHV-1 along with treatment of either DMSO or 2 μM KRN5 during virus infection. After infection for 24 h, cell lysates were prepared and subjected to Western blot to detect viral protein gC. Data represent the means of three independent experiments. Significance was assessed with Student's t test (*, P < 0.05).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Control, Concentration Assay, Virus, Isolation, Quantitative RT-PCR, Western Blot

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of KRN5 had on NFAT5 protein expression in MDBK cells. (A) MDBK cells in 6-well plates were treated with either DMSO control or KRN5 at indicated concentrations. After treatment for 24 h, the cell lysates were prepared and subjected to Western blot to detect the protein levels of NFAT5. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 100%. Data represent the means of three independent experiments. Significance was assessed with Student's t test (ns, not significant).

Article Snippet: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Control, Western Blot, Software

Journal: Molecular cell

Article Title: A consensus binding motif for the PP4 protein phosphatase

doi: 10.1016/j.molcel.2019.08.029

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-GAPDH , Santa Cruz , sc-25778; RRID: AB_10167668.

Techniques: Virus, Recombinant, Protease Inhibitor, Plasmid Preparation, Purification, DNA Ligation, Mutagenesis, Software