Journal: Cell reports

Article Title: Genetic and Functional Dissection of the Role of Individual 5-HT 2 Receptors as Entry Receptors for JC Polyomavirus

doi: 10.1016/j.celrep.2019.04.067

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-5-HT 2B R was obtained from Novus Biologicals cat# NLS1111 and recognizes the second extracellular loop.

Techniques: Generated, Virus, Recombinant, Mutagenesis, Luciferase, Control, Plasmid Preparation, Software, CRISPR

Journal:

Article Title: Vascular Endothelial Growth Factor-A stimulates Snail expression in breast tumor cells: Implications for tumor progression

doi: 10.1016/j.yexcr.2008.05.004

Figure Lengend Snippet: A. MDA-MB-435 and T47D cells were incubated with a NP-1-neutralizing antibody (Anti-NP-1) or an isotype control antibody (IgG) at a concentration of 10μg/ml. After 4 hours, RNA was extracted, and Snail, E-cadherin and β-actin mRNA levels were measured as described in A. B. SUM159 breast tumor cells were transfected with 100nM of a control or VEGF-A siRNA. Four hours after transfection, equivalent amounts of total cellular proteins extracted from these cells were subjected to SDS-PAGE and immunoblotted with a phospho-GSK-3β or total GSK-3β antibody. C & D. MDA-MB-435 cells were incubated with the VEGF-A-neutralizing antibody Avastin (Anti-VEGF), a NP-1-neutralizing (Anti-NP-1) antibody, or an isotype control antibody (IgG) in the presence of DMSO (1:1000) or a small molecule GSK-3 inhibitor (SB415286, 25 μM). After 4 hours, RNA was extracted, and Snail and β-actin levels were determined by reverse-transcription PCR. Similar results for A–D were observed in 3 trials.

Article Snippet: The following antibodies were used in these studies: Neuropilin-1-neutralizing antibody (R&D Systems-MAB566), mouse IgG 2B , (R&D Systems), Avastin ® (Genentech)(kindly provided by Dr. Andrew Nixon, Duke University Medical Center), human IgG1 κ (Sigma), mouse anti E-cadherin (R&D Systems MAB1838), mouse anti Snail [ 12 ], mouse anti β-actin (Sigma), rabbit anti phospho-GSK-3β(Ser9) (Cell Signaling), mouse anti GSK-3 (Upstate), IRDye ™ 700 Donkey anti Mouse IgG and IRDye ™ 800 Goat anti Rabbit IgG (Rockland).

Techniques: Incubation, Concentration Assay, Transfection, SDS Page

Journal:

Article Title: Vascular Endothelial Growth Factor-A stimulates Snail expression in breast tumor cells: Implications for tumor progression

doi: 10.1016/j.yexcr.2008.05.004

Figure Lengend Snippet: MDA-MB-435 and SUM159 cells were incubated with Avastin®; or an isotype control antibody (IgG) at a concentration of 5 μg/mL. A. After 4 hours, RNA was extracted from these cells, and Snail and β-actin levels were assessed by reverse-transcription PCR. B. Total cellular proteins were extracted from SUM159 cells incubated for 4 hours with the indicated antibodies. Equivalent amounts of protein from these cels were subjected to SDS-PAGE and immunoblotted with Snail or β-actin antibodies. Similar results were obtained in 3 independent trials.

Article Snippet: The following antibodies were used in these studies: Neuropilin-1-neutralizing antibody (R&D Systems-MAB566), mouse IgG 2B , (R&D Systems), Avastin ® (Genentech)(kindly provided by Dr. Andrew Nixon, Duke University Medical Center), human IgG1 κ (Sigma), mouse anti E-cadherin (R&D Systems MAB1838), mouse anti Snail [ 12 ], mouse anti β-actin (Sigma), rabbit anti phospho-GSK-3β(Ser9) (Cell Signaling), mouse anti GSK-3 (Upstate), IRDye ™ 700 Donkey anti Mouse IgG and IRDye ™ 800 Goat anti Rabbit IgG (Rockland).

Techniques: Incubation, Concentration Assay, SDS Page

Journal: PLoS ONE

Article Title: De novo Generation of Cells within Human Nurse Macrophages and Consequences following HIV-1 Infection

doi: 10.1371/journal.pone.0040139

Figure Lengend Snippet: (A) Flow cytometry demonstrating that SMC-derived maturing macrophages express CD10 with increasing frequency. Percentages reflect portions of maturing macrophages expressing CD10. Corresponding CD10 versus side scatter (SSC) plots with gates are shown on the left. See for kinetics of expression. (B) Confocal microscopy images demonstrating CD10 expression on a young, EDTA-recovered nurse macrophage at D3 of IL-2 exposure. Size bar: 20 µm. (C) Expression of CD10 in macrophages and CD3+ cells recovered from primary macrophage cultures, as determined by real-time PCR. CD3+ cells were purified from the nonadherent populations using CD3+ magnetic beads. Macrophages were harvested using accutase. For each donor, the mean values obtained from uncultured CD3+ cells were subtracted from the mean values shown for CD3+ cells at each time point, and similarly, the mean values obtained from uncultured CD14+ cells were subtracted from the mean values obtained for the cultured macrophages at each time point. No values are shown for CD3+ cells for donor 8892 at days 17, 21 and 24, because too few nonadherent cells were recovered to permit CD3+ magnetic bead selection. The average percent difference between replicate reactions (including those for GAPDH) was 0.6% (donor 4342) and 0.7% (donor 8892).

Article Snippet: The primary mouse monoclonal antibodies used were: anti-CD3 (clone PS1, IgG2a, Novocastra, Ltd, distributed by Vector Laboratories), anti-CD10 (clone HI10A, IgG 1 , BD Biosciences), anti-CD11b (clone 238446, IgG 2b , R & D Systems), anti-CD36 (185-1G2, IgG 2a , Santa Cruz Biotechnology), anti-CD68 (KP1, IgG 1 Novocastra), anti-MHC II (anti-human HLA-DP/DQ/DR clone CR3/43, IgG 1 , DAKO), anti-HIV-1p24 (Kal-1, IgG 1 , DAKO), anti-α-tubulin (236-10501, IgG1, Molecular Probes) and anti-cdc6 (37F4, IgG2a, Molecular Probes).

Techniques: Flow Cytometry, Derivative Assay, Expressing, Confocal Microscopy, Real-time Polymerase Chain Reaction, Purification, Magnetic Beads, Cell Culture, Selection

Journal: Journal of Virology

Article Title: Flavivirus Infection Impairs Peroxisome Biogenesis and Early Antiviral Signaling

doi: 10.1128/JVI.01365-15

Figure Lengend Snippet: Flavivirus infection results in loss of the peroxisomal matrix enzyme, catalase. (A and B) A549 cells were infected with DENV or WNV (MOI = 1); cell lysates were harvested 16, 24, and 48 h postinfection; and the levels of the peroxisomal and mitochondrial matrix proteins, catalase (A) and Hsp60 (B), respectively, were determined by immunoblotting (left) and quantified (right). The error bars represent standard errors of the mean. *, P < 0.05. (C) Peroxisome size is not affected by flavivirus infection. A549 cells were infected (MOI = 1) with either DENV or WNV. At 48 h postinfection, the cells were processed for indirect immunofluorescence microscopy. Peroxisomes were detected with rabbit polyclonal antibodies to SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 568, and infected cells were identified using human anti-DENV E antibodies or a mouse monoclonal antibody to the WNV NS2B-NS3 complex. Primary antibodies were detected with goat anti-human IgG conjugated to Alexa Fluor 488 or donkey anti-mouse IgG conjugated to Alexa Fluor 488. The images were acquired and reconstructed using a DeltaVision OMX structured-illumination microscope. Volocity software was used to determine the sizes and numbers of peroxisomes in mock-infected and flavivirus-infected cells.

Article Snippet: Rabbit antibodies against FLAG epitope (Sigma), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam), and Pex19 (Epitomic and Abcam) and mouse monoclonal antibodies to GAPDH (Proteintech), Hsp60 (BD Sciences), p32/gC1q-R (Babco, Richmond, CA, and our laboratory [ 13 ]), Lamp1 (Santa-Cruz Biotechnology), PMP70 (Sigma), and WNV NS3/2b (R&D Systems) were purchased from the indicated suppliers.

Techniques: Infection, Western Blot, Immunofluorescence, Microscopy, Software