Journal:

Article Title: Interaction of the Human Immunodeficiency Virus Type 1 Vpr Protein with the Nuclear Pore Complex

doi:

Figure Lengend Snippet: Subcellular localization of β-gal–Vpr fusion proteins in HeLa cells. Monolayers were transfected with wild-type (A) or A30P mutant (C) expression vectors and analyzed by indirect immunofluorescence with a β-galactosidase-specific antiserum. The phase-contrast analyses are also shown (B and D).

Article Snippet: For single-label analyses, the Vpr-Myc, MBP-Vpr, and β-gal–Vpr proteins were detected, respectively, with the Myc-specific monoclonal antibody 9E10 ( 19 ), a rabbit polyclonal anti-MBP antiserum (U.S. Biochemical), and a rabbit polyclonal anti-β-galactosidase antiserum (Organon Teknika).

Techniques: Transfection, Mutagenesis, Expressing, Immunofluorescence

Journal:

Article Title: Interaction of the Human Immunodeficiency Virus Type 1 Vpr Protein with the Nuclear Pore Complex

doi:

Figure Lengend Snippet: Colocalization of wild-type β-gal–Vpr and importin-β at NPCs. Transfected HeLa cells were subjected to double-label immunofluorescence with primary antibodies specific for β-galactosidase (A) or β-importin (B) and analyzed by laser-scanning confocal microscopy. The superimposed images are shown (dual [C]) together with the corresponding differential interference contrast image (DIC [D]).

Article Snippet: For single-label analyses, the Vpr-Myc, MBP-Vpr, and β-gal–Vpr proteins were detected, respectively, with the Myc-specific monoclonal antibody 9E10 ( 19 ), a rabbit polyclonal anti-MBP antiserum (U.S. Biochemical), and a rabbit polyclonal anti-β-galactosidase antiserum (Organon Teknika).

Techniques: Transfection, Immunofluorescence, Confocal Microscopy

Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: Physiological Reports

Article Title: Tuberous sclerosis complex exhibits a new renal cystogenic mechanism

doi: 10.14814/phy2.13983

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: Rabbit polyclonal Anti‐NCC , 1:60 , Stressmarq Biosciences; Victoria, BC, Canada , SPC‐402.

Techniques: Plasmid Preparation, Generated

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Both PCV2 and ORF5 reduced the expression of GPNMB in PAMs. (A) PCR detection of PCV2 nucleic acid in PAM cells with PCV2 infection at a MOI of 1.0 at 24 and 48 h post-infection. Marker. DNA Marker DL1000. (B) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (C) Immunoblot analysis of GPNMB protein expression in PAM cells infected with PCV2 a MOI of 1 at indicated time points. (D) Immunoblot analysis of GFP protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at 48 h post-transfection. (E) Real-time qRT-PCR analysis of GPNMB mRNA expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. (F) Immunoblot analysis of GPNMB protein expression in PAM cells transfected with GFP-C1 or GFP ORF5 construct at indicated time points. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in (B) and (E) . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Infection, Marker, Quantitative RT-PCR, Western Blot, Transfection, Construct

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Interaction of ORF5 with GPNMB. (A) Exogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were co-transfected with plasmids GFP-ORF5 and Flag-GPNMB. PAMs co-transfected with GFP-C1 and Flag-GPNMB were used as negative controls. A quarter of the cell extract was subjected to the input assay to assess β-actin, Flag-fusion and GFP-fusion protein levels. The rest of the extract was subjected to IP assay. Western blot detected proteins with a mouse anti-GFP mAb and a mouse anti-Flag pAb. (B) ORF5 protein co-localizes with GPNMB. HEK-293T cells were co-transfected with pGPNMB-Red and pORF5-GFP, pEGFP-C1 and pDsRed-N1 were used as control. Cells were fixed and stained with DAPI (blue) at 48 h post-transfection. Scale bar = 10 μm. (C) Endogenous co-IP analysis of ORF5 and GPNMB in PAMs. Cells were transfected with plasmid GFP-ORF5-Flag and GFP-C1-transfected PAMs were used as negative controls. The input assay was performed using a quarter of the cell extract to assess β-actin, Flag fusion protein and GPNMB levels. (D) GST-ORF5 pull-down assay. The GST and GST-ORF5 proteins expressed in Escherichia coli Rosetta (DE3) cells were immobilized on a glutathione agarose resin, followed by incubation of the resin with the cell lysates containing GPNMB-Flag protein.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot, Staining, Plasmid Preparation, Pull Down Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Stable overexpression of GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells with stable GPNMB overexpression. (B) Immunoblot analysis of GPNMB protein in PK-15 cells with stable GPNMB overexpression. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells with stable GPNMB overexpression. Different cell lines were infected with PCV2 at a MOI of 0.1. PCV2 viral RNA level was measured 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Western Blot, Infection

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Transient overexpression of Red-fused GPNMB inhibits PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. (B) Immunoblot analysis of GPNMB protein in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1 at 24 h post-transfection. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in PK-15 cells transfected with pDsRed-GPNMB or PdsRed-N1. Different cells were infected with PCV2 at a MOI of 0.1 at 24 h post-transfection. RNA expression level was measured at 24 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB increases PCV2 replication and ORF5 expression. (A) Real-time qRT-PCR analysis of GPNMB mRNA expression in stable GPNMB knockdown PK-15 cells. PK-15 cells transduced with GPNMB-knockdown lentivirus (shGPNMB-1, 2, 3) or random sequence control (shN). (B) Immunoblot analysis of GPNMB protein in stable GPNMB knockdown PK-15 cells. The right panel is the GPNMB protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (C,D) Real-time qRT-PCR analysis of PCV2 viral RNA (C) and ORF5 mRNA (D) expression in stable GPNMB knockdown. Different cells were infected with PCV2 at a MOI of 0.1. RNA expression level was measured at 24 h or 48 h post-infection. Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A , C , and D . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Expressing, Quantitative RT-PCR, Transduction, Sequencing, Western Blot, Infection, RNA Expression

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Overexpression of GPNMB promotes cell cycle progression. (A) Real-time qRT-PCR analysis of Cyclin A mRNA expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (B) Immunoblot analysis of Cyclin A protein expression in PAM cells transfected with pEGFP-C1 or pEGFP-ORF5 construct at 24 h or 48 h post-transfection. (C,D) Real-time qRT-PCR analysis of GPNMB (C) and Cyclin A (D) mRNA expression in PAMs with stable GPNMB overexpression. (E) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with stable GPNMB overexpression. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (F) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (Lenti) and GPNMB overexpression lentivirus transduced cells (Lenti-GPNMB). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Construct, Western Blot, Flow Cytometry, Staining

Journal: Frontiers in Microbiology

Article Title: A Host Factor GPNMB Restricts Porcine Circovirus Type 2 (PCV2) Replication and Interacts With PCV2 ORF5 Protein

doi: 10.3389/fmicb.2018.03295

Figure Lengend Snippet: Knockdown of GPNMB inhibits cell cycle progression. (A,B) Real-time qRT-PCR analysis of GPNMB mRNA (A) and Cyclin A mRNA (B) expression in PAMs cell lines with GPNMB knockdown. (C) Immunoblot analysis of GPNMB and Cyclin A protein levels in PAM cells with lines with GPNMB knockdown. The middle panel is the GPNMB protein level and the right panel is the Cyclin A protein level that the intensity of the signal for targeted protein were normalized to that from β-actin with three independent experiments. (D) Histograms from flow cytometry data for propidium iodide (PI) staining for the mock cells (Mock), control lentivirus transduced cells (shN) and GPNMB knockdown lentivirus transduced cells (shGPNMB-3). Data are shown as the mean ± SD of three independent experiments and measured in technical duplicates in panels A and B . Data were normalized to housekeeping gene β-actin expression. Comparisons between groups were determined with the Student’s t -test. ∗ p < 0.05.

Article Snippet: Membranes were blocked in TBST, a blocking buffer containing 5% skim milk for 2 h at room temperature, followed by incubation with primary antibodies, including rabbit anti-GPNMB polyclonal antibody, rabbit anti-Cyclin A polyclonal antibody (1:1000, Santa Cruz Biotechnology, United States), and rabbit anti-β-actin polyclonal antibody (1:2000, CWBIO, China) at 4°C for overnight.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Staining

Journal: Stem Cell Reports

Article Title: Androgen Maintains Intestinal Homeostasis by Inhibiting BMP Signaling via Intestinal Stromal Cells

doi: 10.1016/j.stemcr.2020.08.001

Figure Lengend Snippet: The Effect of Androgen on ISC Proliferation For counting of BrdU + cells, 10 crypts were randomly chosen for investigation. (A) For detection of BrdU + cells per crypt in the ORX model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm. (B) For detection of BrdU + cells per crypt in the inhibition of AR model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm. (C) For detection of BrdU + cells per crypt in the OVX model, IHC was performed with primary antibody to BrdU. Scale bar, 100 μm. (D) For analysis of active β-CATENIN and total β-CATENIN expression, western blotting was performed. (E) For detection of intestinal stem cells in the ORX model, IHC was performed with primary antibody to Olfm4 + . Scale bar, 50 μm. (F) For analysis of relative mRNA levels of Bmp4 , quantitative real-time PCR was performed in the ORX, OVX, and inhibition of AR models. (G) The phosphorylation of Smad1/5 was assessed by western blot analysis. Data, indicated as mean ± SD, correspond to three independent experiments (A, C, n = 6; B, n = 5; D, G, n = 2; E, F, n = 4 mice/group/experiment). ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: The primary antibodies were anti-β-catenin (BD Biosciences, lot 610154), anti-active β-catenin (Cell Signaling, lot D13A1), anti-psmad1/5 (Cell Signaling, lot 41D10); and anti-math1 (Santa Cruz, lot sc-136173).

Techniques: Inhibition, Expressing, Western Blot, Real-time Polymerase Chain Reaction