Journal: Protein & Cell

Article Title: The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

doi: 10.1007/s13238-020-00764-0

Figure Lengend Snippet: NP L692A , NP P697A , NP P698A , and NP W699A inhibit the migration of VP40 to the plasma membrane . (A) The kinetic progress of IB migration to the PM. HeLa cells were cultured on 20 mm dishes and co-transfected with mCherry-VP40 and GFP-NP or GFP-NP W699A , together with their respective functional proteins, to exclude the effect of fluorescence tags. Live-cell imaging analysis was started at 24 h post-transfection. The pictures show the maximum intensity projection of time-lapse images of the cells. Images were captured every 120 s. Scale bar = 5 μm. (B and C) Isolation of cytosol and plasma membrane (PM) fractions. HEK293T cells were transfected with the indicated plasmids for 24 h. The cytosol and PM fractions were isolated and then subjected to a WB assay. GAPDH and Na/K ATPase served as controls for the cytosol or the PM fraction, respectively (B); quantification of the amount of PM-associated VP40 is shown (C). (D and E) The impact of NP P697A on the interaction of VP40 with Sec24C. HEK293T cells were co-transfected with VP40-Flag, HA-Sec24C, and Myc-NP/NP P697A for 30 h. IP was performed with anti-HA antibodies (D); quantification of the ability of VP40 binding Sec24C in the presence of NP/NP P697A is shown (E). Images are representative of three independent experiments. Error bars, mean ± SD of three independent experiments ( n = 3). Student’s t -test; * P < 0.05; ** P < 0.01; *** P < 0.001; ns, non-significant

Article Snippet: The membrane was blocked with 5% skim milk powder in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST) for 30 min before being incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Fisher Scientific) for 1 h. The primary antibodies were used as follows: mouse anti-Myc (1:10,000, MBL), mouse anti-HA (1:10,000, MBL), mouse anti-Flag (1:10,000, Sigma), rabbit anti-HA (1:10,000, Sigma), rabbit anti-Flag (1:10,000, CST), rabbit anti-Myc (1:10,000, Sigma), rabbit anti-VP30 pSer29 antibody (1:1000, ABclonal), rabbit anti-Na/K ATPase (1:1000, ABclonal), and mouse anti-GAPDH (1:1000, ABclonal).

Techniques: Migration, Cell Culture, Transfection, Functional Assay, Fluorescence, Live Cell Imaging, Isolation, Binding Assay

Journal: BMC Neuroscience

Article Title: Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

doi: 10.1186/1471-2202-6-7

Figure Lengend Snippet: Reduced activity and protein levels of the Na+/K+ ATPase enzyme in APP+PS1 mice. Activity of total (A) and ouabain-sensitive (B) ATPase was assayed colorimetrically in APP+PS1 mice tissue (n = 8, open bars) and non-transgenic littermate tissue (n = 8, solid bars), presented here as μmols of phosphate liberated by ATPase per mg of protein per hour. In the amyloid-containing hippocampus, there was a significant decrease in the specific activity of ouabain-sensitive ATPase in APP+PS1 mice compared to non-transgenic mice, but there was no decrease in cerebellar Na+/K+ ATPase activity. Cerebellar activity was 20% of that seen in non-transgenic hippocampus. Panel C shows the quantitation of the optical density of bands corresponding to the molecular weight of Na+/K+ ATPase using standard Western blot technique. This reveals a trend for decreased protein levels. *** indicates significant differences between APP+PS1 mice and non-transgenic littermates (p < 0.001) when measured by one-way ANOVA.

Article Snippet: Sections were washed the following day and co-incubated with a 1:5000 dilution of a rabbit anti-rat Na+/K+ ATPase αIII IgG (Upstate Biotech, Lake Placid, NY) and a 1:10,000 dilution of a mouse monoclonal IgG1 ascites pool specific for phosphorylated forms of neurofilament (SMI-312; Sternberger Monoclonals, Lutherville, MD) in 2% goat serum and 2% horse serum in PBS.

Techniques: Activity Assay, Transgenic Assay, Quantitation Assay, Molecular Weight, Western Blot

Journal: BMC Neuroscience

Article Title: Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

doi: 10.1186/1471-2202-6-7

Figure Lengend Snippet: Verification of Na+/K+ ATPase αIII antibody specificity by immunohistochemistry following pre-incubation with purified protein and Western blotting . Horizontal sections were immunostained for Na+/K+ ATPase αIII with (Panel B) and without (Panel A) pre-incubation with 70 μunits of purified enzyme followed by Congo red staining. The pre-incubation significantly decreased staining, confirming antibody specificity. Scale bar = 50 μm.

Article Snippet: Sections were washed the following day and co-incubated with a 1:5000 dilution of a rabbit anti-rat Na+/K+ ATPase αIII IgG (Upstate Biotech, Lake Placid, NY) and a 1:10,000 dilution of a mouse monoclonal IgG1 ascites pool specific for phosphorylated forms of neurofilament (SMI-312; Sternberger Monoclonals, Lutherville, MD) in 2% goat serum and 2% horse serum in PBS.

Techniques: Immunohistochemistry, Incubation, Purification, Western Blot, Staining

Journal: BMC Neuroscience

Article Title: Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

doi: 10.1186/1471-2202-6-7

Figure Lengend Snippet: Hippocampal and cortical immunohistochemistry for Na+/K+ ATPase in APP+PS1 mice and non-transgenic littermates . Horizontal hippocampal and cortical sections were immunostained for Na+/K+ ATPase αII. The left panels depict hippocampus, while the right panels encompass the cerebral cortex. Panels A & B: high power magnification (Scale bar = 8.33 μm) revealed membrane-localized staining for Na+/K+ ATPase in the insular cortex (Panel B) and CA3 of the hippocampus (Panel A) of non-transgenic mice. Panel C: hippocampal staining of non-transgenic mice showed a ubiquitous distribution of Na+/K+ ATPase throughout the neuropil, while less staining was apparent along the pyramidal layer of Ammon's horn, the hilus and the granular layer of the dentate gyrus. Panel D: cortical staining was also substantial with slight lightening in layers 1 and 2. White matter in both structures remained unstained. A similar staining pattern was observed in the APP+PS1 mice with the exception of focal non-stained areas in the gray matter, presumably where amyloid plaques are located (Panel E [hippocampus] & F [cortex]). Immunostaining followed by Congo red histochemistry confirmed that the Na+/K+ ATPase staining was absent not only immediately where amyloid was located, but also in a zone surrounding the plaque (Panels G [hippocampus] & H [cortex]). Scale bar for panels C-H = 120 μm.

Article Snippet: Sections were washed the following day and co-incubated with a 1:5000 dilution of a rabbit anti-rat Na+/K+ ATPase αIII IgG (Upstate Biotech, Lake Placid, NY) and a 1:10,000 dilution of a mouse monoclonal IgG1 ascites pool specific for phosphorylated forms of neurofilament (SMI-312; Sternberger Monoclonals, Lutherville, MD) in 2% goat serum and 2% horse serum in PBS.

Techniques: Immunohistochemistry, Transgenic Assay, Staining, Immunostaining

Journal: BMC Neuroscience

Article Title: Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

doi: 10.1186/1471-2202-6-7

Figure Lengend Snippet: Dual Immunostaining of Na+/K+ ATPase αIII and dystrophic neurites . Horizontal sections were immunostained for Na+/K+ ATPase using DAB with nickel intensification followed by Congo red staining for Aβ plaques. A discernable circumferential void in ATPase staining surrounding the plaque was observed (Panel A, scale bar = 50 μm; Panel B, scale bar = 16.67 μm). Immunofluorescent staining of dystrophic neurites using the anti-phosphorylated neurofilament antibody SMI-312 followed by Congo red staining demonstrate a close relationship between amyloid plaques and dystrophic neurites (Panel C; white arrows demarcate the neurites). Congo red yellow fluorescence was digitally suppressed to more clearly reveal the green-stained neurites (scale bar = 16.67 μm). Panel D is a digital overlay of the fluorescein-labeled dystrophic neurite image onto the bright field peroxidase-labeled Na+/K+ ATPase + Congo red image which demonstrates that dystrophic neurites are present predominantly in the zone devoid of Na+/K+ ATPase staining surrounding the congophilic plaques (scale bar = 16.67 μm).

Article Snippet: Sections were washed the following day and co-incubated with a 1:5000 dilution of a rabbit anti-rat Na+/K+ ATPase αIII IgG (Upstate Biotech, Lake Placid, NY) and a 1:10,000 dilution of a mouse monoclonal IgG1 ascites pool specific for phosphorylated forms of neurofilament (SMI-312; Sternberger Monoclonals, Lutherville, MD) in 2% goat serum and 2% horse serum in PBS.

Techniques: Immunostaining, Staining, Fluorescence, Labeling

Journal: BMC Neuroscience

Article Title: Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

doi: 10.1186/1471-2202-6-7

Figure Lengend Snippet: Amyloid-beta 1–42 peptide inhibits Na+/K+ ATPase activity . Purified cerebral Na+/K+ ATPase was pre-incubated with vehicle or Aβ 1–42, and enzyme activity percentages of the vehicle (20 μmols Pi/mg protein/hour) are presented (mean ± SEM). "◊" indicates the values for the soluble (DMSO+water) Aβ suspension. "○" indicates the values for the fibrillar (DMSO+neutralized HCl) Aβ suspension. At the highest concentrations, the soluble preparation dramatically reduces Na+/K+ ATPase activity compared to both vehicle and the fibrillar Aβ preparation. * (p < 0.05) and *** (p < 0.001) indicates significant differences between Aβ and vehicle alone when measured by one-way ANOVA.

Article Snippet: Sections were washed the following day and co-incubated with a 1:5000 dilution of a rabbit anti-rat Na+/K+ ATPase αIII IgG (Upstate Biotech, Lake Placid, NY) and a 1:10,000 dilution of a mouse monoclonal IgG1 ascites pool specific for phosphorylated forms of neurofilament (SMI-312; Sternberger Monoclonals, Lutherville, MD) in 2% goat serum and 2% horse serum in PBS.

Techniques: Activity Assay, Purification, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Enhanced recruitment of glutamate receptors underlies excitotoxicity of mitral cells in acute hyperammonemia

doi: 10.3389/fncel.2022.1002671

Figure Lengend Snippet: Blocking of exocytosis with TAT-NSF700 or blocking dynamin-dependent endocytosis changes the effect of NH 4 + on the excitability of MCs. (A) Example traces of sEPSCs from a MC in the presence of Bic + Stry, showing that the sEPSC amplitude, integral area, and frequency were unchanged by NH 4 Cl after OB slices were pre-treated with TAT-NSF700 (5 μM, 30 min), a permeable thrombin-induced exocytosis inhibitor. (B–D) Post hoc statistical analysis of sEPSC amplitude, integral area, and frequency in Ctrl and NH 4 Cl solution after OB slices were pre-incubated with TAT-NSF700. (E) Typical sEPSC recordings from a MC in Ctrl (Bic + Stry in aCSF) and Ctrl + NH 4 Cl buffer with OB slices pre-incubated with dynasore (40 μM, 30 min), an inhibitor for dynamin-dependent endocytosis. NH 4 Cl effectively augmented sEPSC amplitude and integral area. (F–H ) Mean sEPSC amplitude, integral area, and frequency in Ctrl (Bic + Stry in aCSF) and NH 4 Cl solution in OB slices pre-incubated with dynasore. (I) Representative western blotting images of NR1, GluR1 and Na/K-ATPase from OB in the treatment of aCSF (Ctrl) and NH 4 Cl, respectively. (J) Qualification of intensity of NR1 and GluR1 normalized to Ctrl (aCSF). Error bars represent standard error; * p < 0.05, ** p < 0.01; ns, not significant; paired Student’s t -test, unpaired Student’s t -test.

Article Snippet: Separated proteins were transferred onto a NC membrane, blocked in 5% non-fat dry milk for at least 1 h at room temperature and subsequently incubated overnight at 4°C on a shaker with primary antibody against rabbit anti-NR1, rabbit anti-GluR1 (purchased from abclonal, Wuhan, China, 1:1000 dilution) and rabbit anti-Na/K-ATPase (Servicebio, Wuhan, China, 1:1000 dilution).

Techniques: Blocking Assay, Incubation, Western Blot