Journal: PLoS Pathogens

Article Title: Rotavirus NSP1 Inhibits NFκB Activation by Inducing Proteasome-Dependent Degradation of β-TrCP: A Novel Mechanism of IFN Antagonism

doi: 10.1371/journal.ppat.1000280

Figure Lengend Snippet: (A) MA104 cells were infected with three pfu/cell of the indicated virus strain. Lysates were prepared at 2, 4, 6, 8, and 10 hpi and the abundance of p65 was determined by immunoblot using anti-p65 antibody. Blots were probed with anti-GAPDH as a loading control. (B) The subcellular localization of p65 in MA104 cells infected with three pfu/cell of the indicated virus strain was determined at six hpi by confocal microscopy (63×, NA 1.40). Cells were stained with anti-p65 antibody, followed by Alexa Fluor 594-conjugated goat anti-rabbit IgG.

Article Snippet: The primary antibodies were mouse anti-IRF3 (Santa Cruz Biotechnology) or rabbit anti-p65 (Rockland).

Techniques: Infection, Virus, Western Blot, Control, Confocal Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Rotavirus NSP1 Inhibits NFκB Activation by Inducing Proteasome-Dependent Degradation of β-TrCP: A Novel Mechanism of IFN Antagonism

doi: 10.1371/journal.ppat.1000280

Figure Lengend Snippet: MA104 cells were infected with A5-16, OSU or NCDV at an moi of three pfu/cell. Six hours post-infection, nuclear and cytoplasmic fractions were separated with the nuclear extract kit following the manufacturer's instructions (Active Motif). (A) p65 activation measured by p65 TransAm ELISA. Error bars are the standard error of the mean. (B) Nuclear fractions were probed with anti-p65 antibody, anti-laminA/C (nuclear, BD Biosciences), and anti- GAPDH (cytoplasmic) antibodies.

Article Snippet: The primary antibodies were mouse anti-IRF3 (Santa Cruz Biotechnology) or rabbit anti-p65 (Rockland).

Techniques: Infection, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet: Morphological changes in the cytoskeleton of dysfunctional VSMCs Scale bar: 20 μm

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques:

Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet: Schematic workflow supernatant assay Immunofluorescence staining shows microglia activation after incubation with supernatant from dysfunctional VSMCs. Activated microglial were characterized by morphologic changes and elevated expression of IL-6 at 100 × magnification, scale bars: 5 μm

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques: Immunofluorescence, Staining, Activation Assay, Incubation, Expressing

Journal: STAR Protocols

Article Title: Protocol to assess the effects of dysfunctional human vascular smooth muscle cells on other brain cells using in vitro models of Alzheimer’s disease

doi: 10.1016/j.xpro.2022.101149

Figure Lengend Snippet:

Article Snippet: Culture medium ready-to-use with FBS and antibiotics (VSMCs) , Cell Applications , Cat#311-500.

Techniques: Recombinant, Wound Healing Assay, Staining, SYBR Green Assay, Software, Real-time Polymerase Chain Reaction, Microscopy, Sterility, Inverted Microscopy, Confocal Microscopy

Journal: Scientific Reports

Article Title: Application across species of a one health approach to liquid sample handling for respiratory based -omics analysis

doi: 10.1038/s41598-021-93839-9

Figure Lengend Snippet: Analysis of tracheal wash derived macrophages. Flow cytometry results showing cross-reactivity of mouse anti-human CD163 antibody against equine tracheal macrophages. (a) Isotype control, (b) CD163 stained cells (c) overlay of CD163+ population on top of total cells. (d) Leishman stained cytospin preparations of CD163+ cells by light microscopy (× 20, scale bar = 50 μm). Data and image analysis was performed in FlowJo ® v10.5.3 https://www.flowjo.com/ .

Article Snippet: Briefly, the cells were first stained with a R-Phycoerythrin (PE)-conjugated CD163 primary antibody; subsequently, the cells were magnetically labelled with anti-PE MicroBeads (Miltenyi Biotec, cat no 130-105-639) before the cell suspension was loaded on a MACS ® LS Column (Miltenyi Biotec Ltd., cat n0 130-042-401), placed in the magnetic field of a MACS Separator.

Techniques: Derivative Assay, Flow Cytometry, Control, Staining, Light Microscopy

Journal: Cell Death & Disease

Article Title: Pharmacological antagonism of kainate receptor rescues dysfunction and loss of dopamine neurons in a mouse model of human parkin-induced toxicity

doi: 10.1038/s41419-020-03172-8

Figure Lengend Snippet: Representative images showing TH immunoperoxidase labeling in the SNc of the WT and the parkinQ311X mice (6 months of age) treated with either vehicle or UBP310 (20 mg/kg i.p.) for 90 or 135 days. The graph below the picture shows DA neuron quantification performed by stereological count. Data are the means ± SEM of TH-positive neurons: WT treated with vehicle for 90 days 5578 ± 235 n = 8 mice, WT treated with UBP310 for 90 days 5351 ± 162 n = 7 mice, parkinQ311X treated with vehicle for 90 days 4701 ± 117 n = 11 mice, parkinQ311X treated with UBP310 for 90 days 5347 ± 107 n = 12 mice, parkinQ311X mice treated with UBP310 for 135 days 5514 ± 94 n = 9 mice, one-way ANOVA followed by Tukey’s test F = 6.681, WT vehicle 90 days vs. parkinQ311X vehicle 90 days *** p = 0.0007, WT UBP310 90 days vs. parkinQ311X vehicle 90 days * p = 0.0260, parkinQ311X vehicle 90 days vs. parkinQ311X UBP310 90 days ** p = 0.0073, parkinQ311X vehicle 90 days vs. parkinQ311X UBP310 135 days ** p = 0.0012.

Article Snippet: UBP310 (3-({3-[(2S)-2-Amino-2-carboxyethyl]-5-methyl-2,6-dioxo-3,6-dihydro-1(2H)-pyrimidinyl}methyl)-2-thiophenecarboxylic acid) was purchased from TOCRIS (Bristol, UK), dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL, and then diluted in 90% saline + 10% DMSO at 1 mg/mL.

Techniques: Labeling

Journal: Cell Death & Disease

Article Title: Pharmacological antagonism of kainate receptor rescues dysfunction and loss of dopamine neurons in a mouse model of human parkin-induced toxicity

doi: 10.1038/s41419-020-03172-8

Figure Lengend Snippet: a Examples of patch-clamp whole-cell recordings in current-clamp mode from an SNc DA neuron in an acute slice of ventral midbrain. The upper traces show membrane potential hyperpolarizing and depolarizing responses to intracellular injection of negative and positive current steps, respectively (±200 pA, 500 ms, lower traces). Note the relatively depolarized resting-membrane potential (−49 mV) and the prominent I h -dependent depolarizing “sag” (arrow) typical of DA neurons. b Cell-attached recordings of spontaneous action potential (AP) firing of SNc DA neurons from WT and parkinQ311X mice at 25 days of age. The dot plot on the right shows the quantification of spontaneous firing frequencies. The mean firing frequency of the SNc DA neurons was significantly higher in parkinQ311X mice compared to WT mice (mean ± SEM WT: 1.75 ± 0.18 Hz, parkinQ311X mice: 2.96 ± 0.23 Hz). Data were obtained from 6 WT mice (16 recorded neurons) and 8 parkinQ311X mice (36 recorded neurons). Unpaired Student t test with Welch correction, *** p = 0.0001, t = 4.148, d f = 47.75. c Summary dot plots showing spontaneous firing frequencies of SNc DA neurons in acute slices of ventral midbrain prepared from WT and parkinQ311X mice at 25 days of age upon extracellular perfusion with UBP310 (5 and 10 µM) and subsequent washout. Mean ± SEM, WT ACSF 2.3 ± 0.46 Hz, WT treated with UBP310 5 µM 2.03 ± 0.37 Hz, WT treated with UBP310 10 µM 1.85 ± 0.32 Hz, WT washout 1.96 ± 0.31 Hz; * p = 0.02, F = 7.83, Friedman repeated-measure ANOVA on ranks. Q311X ACSF 3.4 ± 0.5 Hz, Q311X treated with UBP310 5 µM 2.61 ± 0.36 Hz, Q311X treated with UBP310 10 µM 2.46 ± 0.41 Hz, Q311X washout 3.0 ± 0.55 Hz; one-way repeated-measure ANOVA Holm–Sidak test ** p = 0.009, F = 11.87 (7 recorded neurons for each condition).

Article Snippet: UBP310 (3-({3-[(2S)-2-Amino-2-carboxyethyl]-5-methyl-2,6-dioxo-3,6-dihydro-1(2H)-pyrimidinyl}methyl)-2-thiophenecarboxylic acid) was purchased from TOCRIS (Bristol, UK), dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL, and then diluted in 90% saline + 10% DMSO at 1 mg/mL.

Techniques: Patch Clamp, Membrane, Injection

Journal: Cell Death & Disease

Article Title: Pharmacological antagonism of kainate receptor rescues dysfunction and loss of dopamine neurons in a mouse model of human parkin-induced toxicity

doi: 10.1038/s41419-020-03172-8

Figure Lengend Snippet: a Representative Western blots showing GluK2 immunostaining from total brain lysates obtained from WT or parkinQ311X mice treated with 20 mg/kg UBP310 i.p. for either 4 or 8 h. The graphs show the means ± SEM from densitometer quantifications (WT—white bars, Q311X—gray bars). UBP310 treatment induced a decrease in GluK2 levels. WT mice, NT 1.00 ± 0.02, 4 h 0.77 ± 0.03, 8 h 0.71 ± 0.08, 4 h vs. NT * p = 0.047, 8 h vs. NT * p = 0.0011, F = 7.276, one-way ANOVA with Bonferroni’s test. ParkinQ311X mice, NT 1.00 ± 0.05, 4 h 0.92 ± 0.04, 8 h 0.73 ± 0.02, 8 h vs. NT ** p = 0.0013, F = 13.36, one-way ANOVA with Bonferroni’s test. UBP310 did not change the levels of AMPA receptor subunits GluA1 and GluA2. b Representative confocal images showing TH (red) and GluK2 (green) double immunofluorescence in the SNc of WT or parkinQ311X mice treated with either vehicle or 20 mg/kg UBP310 i.p. for 8 h. Images were acquired using a Leica TCS SP2 confocal microscope, objective 40× (bar, 50 µm). The histograms show the means ± SEM from fluorescence-intensity quantifications. Twelve sections per genotype/treatment were examined derived from n = 3 WT mice treated with vehicle, n = 3 WT mice treated with UBP310, n = 3 parkinQ311X mice treated with vehicle, and n = 3 parkinQ311X mice treated with UBP310. GluK2/TH: WT vehicle 1.00 ± 0.08 n = 95 neurons, WT UBP310 0.7 ± 0.09 n = 91 neurons, parkinQ311X vehicle 1.67 ± 0.08 n = 102 neurons, parkinQ311X UBP310 1.25 ± 0.07 n = 100 neurons, WT vehicle vs. WT UBP310 * p = 0.039, Q311X vehicle vs. Q311X UBP310 p = 0.0006, WT vehicle vs. Q311X vehicle p = 0.00009, F = 27.95, one-way ANOVA followed by Tukey’s test.

Article Snippet: UBP310 (3-({3-[(2S)-2-Amino-2-carboxyethyl]-5-methyl-2,6-dioxo-3,6-dihydro-1(2H)-pyrimidinyl}methyl)-2-thiophenecarboxylic acid) was purchased from TOCRIS (Bristol, UK), dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL, and then diluted in 90% saline + 10% DMSO at 1 mg/mL.

Techniques: Western Blot, Immunostaining, Immunofluorescence, Microscopy, Fluorescence, Derivative Assay

Journal: American journal of physiology. Cell physiology

Article Title: HSP90 modulates the myosin replacement rate in myofibrils.

doi: 10.1152/ajpcell.00245.2017

Figure Lengend Snippet: Fig. 2. Inhibition of heat shock protein 90 (HSP90) activity slows the myosin replacement rate in myofibrils. A–L: sarcomere structures were not disrupted by geldanamycin (GM) treatment. DMSO (A–C and G–I) or 5 g/ml GM (D–F and J–L)-treated cells were stained with indicated antibodies. C, F, I, and L are merged images of A and B, D and E, G and H, and J and K. Arrowheads depict myomesin-positive M-lines in the A-bands in A–F. All images were taken with a confocal microscope. Scale bars 10 m. M–O: relative fluo- rescence intensity after bleaching of green fluorescent protein-tagged myosin heavy chain 3 (GFP-MYH3) was measured in myotubes treated with DMSO or GM. Fluorescence signals were measured at indicated time points (M). Mobile fractions (%) of GFP-MYH3 were 47.0 5.9 in DMSO and 9.6 1.2 in GM (N). Half- lives (hours) of GFP-MYH3 were 6.7 1.2 in DMSO and 3.4 1.0 in GM (O). Values represent the mean SE. *P 0.05, significant difference compared with DMSO control. DMSO control myotubes, n 7; GM-treated myotubes, n 4.

Article Snippet: The antibodies used in this study were mouse anti-myomesin antibody (1:10 dilution; clone B4; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) (19), mouse anti-HSP90 antibody (1:400 dilution; clone D7 ; StressMarq Biosciences), and rabbit polyclonal anti-MYH antibody (1:500 dilution; kindly gifted by the late Prof. Howard Holtzer, University of Pennsylvania, Philadelphia).

Techniques: Inhibition, Activity Assay, Staining, Microscopy, Fluorescence, Control

Journal: Chem Catalysis

Article Title: Selectivity control by modifying pressure, temperature, and ionomer decoration for CO2 electroreduction using gas-diffusion Cu electrodes

doi: 10.1016/j.checat.2024.101030

Figure Lengend Snippet: Figure 1. Characterization of the working electrode (A) TEM images of the as-synthesized Cu composite. (B) SEM image of the as-prepared Cu gas-diffusion electrode (GDE) and inset image show the water contact angle at around 140. (C) In situ Raman spectroscopy monitoring the pre-reduction processes at various applied potentials in 1 M KHCO3 at 298 K. SHE means standard hydrogen electrode. (D) X-ray diffraction patterns of pristine Cu GDE and Cu GDE after reduction together with bare gas-diffusion layer (GDL). (E) XPS analysis of Cu 2p regions of the pristine Cu GDE and Cu GDE after reduction. (F) Auger emission spectra of Cu LMM region of the pristine Cu GDE and Cu GDE after reduction.

Article Snippet: The in situ surface-enhanced Raman spectroscopy was carried out using an inverted confocal Raman microscope (JASCO RMP-510) equipped with an He/Ne laser (532 nm).

Techniques: Synthesized, Diffusion-based Assay, In Situ, Raman Spectroscopy