Journal: bioRxiv

Article Title: Lysosomal Exocytosis Releases Pathogenic α-Synuclein Species from Neurons

doi: 10.1101/2021.04.10.439302

Figure Lengend Snippet: ( a-c ) Recombinant purified myc-αSyn was shaken at 37°C in presence of concentrated extracellular medium from mouse cortical neuron cultures collected over 7 days (DIV 42-49), generated either from wild type (WT) mice, or from Tg x2 -αSyn A53T mice with or without lentiviral expression of VAMP7 dominant-negative fragment (VAMP7 DN ; infected at DIV 7). Aggregation of myc-αSyn was analyzed at the indicated days of incubation by the following assays: ( a ) Congo-red derivative, amyloid-binding dye K114 fluorescence at 390/535 nm (n=4). ( b ) Amyloid-binding dye Thioflavin-T fluorescence at 450/485 nm (n=4). ( c ) Quantitative immunoblotting for the myc epitope-tag, where aggregation is measured as disappearance of monomeric myc-αSyn (top; n=4); dot-blotting for filamentous myc-αSyn aggregates using αSyn Fila antibody (middle; n=4); and dot-blotting for amyloid-type myc-αSyn aggregates using αSyn Amyl A11 antibody (bottom; n=4). All data represent means ± SEM, where each ‘n’ is an independent aggregation experiment. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 by RM 2-way ANOVA.

Article Snippet: β-Actin: Sigma (A1978); A11 oligomers: Stressmarq (SPC-506D); ATP5G: Abcam (ab181243); Calreticulin: Thermo Fisher (OTI15F5) and Novus (NB600-103); Cathepsin-L: Novus (JM10-78); CD81: Novus (SN206-01); EEA1: Thermo Fisher (MA5-14794); Flag: Sigma Cl.

Techniques: Recombinant, Purification, Generated, Expressing, Dominant Negative Mutation, Infection, Incubation, Binding Assay, Fluorescence, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Adapentpronitrile, a New Dipeptidyl Peptidase-IV Inhibitor, Ameliorates Diabetic Neuronal Injury Through Inhibiting Mitochondria-Related Oxidative Stress and Apoptosis

doi: 10.3389/fncel.2018.00214

Figure Lengend Snippet: List of primary and secondary antibodies used in the study.

Article Snippet: Membranes were probed overnight at 4°C with primary antibodies for specific detection of APP (dilution 1:500; Boster, China, BA0581), Aβ (dilution 1:1000; Abcam, United Kingdom, ab62658), GLP-1R (dilution 1:500, Bioss, China, bs-1559R), Bcl-2 (dilution 1:500; Abcam, United Kingdom, ab196495), Bax (dilution 1:400; Proteintech, China, 50599-2-Ig), cytochrome c (dilution 1:1000; Abcam, United Kingdom, ab133504), caspase-9 (dilution 1:1000; Abcam, United Kingdom, ab184786), caspase-3 (dilution 1:1000; Abcam, United Kingdom, ab184787), and β-actin (dilution 1:3000; Proteintech, United States, 60008-1-Ig; Table ).

Techniques:

Journal: Frontiers in Cellular Neuroscience

Article Title: Adapentpronitrile, a New Dipeptidyl Peptidase-IV Inhibitor, Ameliorates Diabetic Neuronal Injury Through Inhibiting Mitochondria-Related Oxidative Stress and Apoptosis

doi: 10.3389/fncel.2018.00214

Figure Lengend Snippet: Effects of adapentpronitrile on pathomorphology and the expression of APP and Aβ proteins in hippocampus and cortex of rat. (A) Representative neuronal pathomorphology in hippocampus and cortex. Sections were pictured at 400×. Scale bar = 50 μm. (B) The expressions of APP and Aβ proteins were measured by Western Blotting. Data were expressed as mean ± SD of four independent experiments, and were analyzed statistically using one-way ANOVA, followed by Dunnett-t type multiple comparison tests. # P < 0.05 and ## P < 0.01 vs. Control group, respectively; ∗ P < 0.05 and ∗∗ P < 0.01 vs. HFD/STZ group, respectively.

Article Snippet: Membranes were probed overnight at 4°C with primary antibodies for specific detection of APP (dilution 1:500; Boster, China, BA0581), Aβ (dilution 1:1000; Abcam, United Kingdom, ab62658), GLP-1R (dilution 1:500, Bioss, China, bs-1559R), Bcl-2 (dilution 1:500; Abcam, United Kingdom, ab196495), Bax (dilution 1:400; Proteintech, China, 50599-2-Ig), cytochrome c (dilution 1:1000; Abcam, United Kingdom, ab133504), caspase-9 (dilution 1:1000; Abcam, United Kingdom, ab184786), caspase-3 (dilution 1:1000; Abcam, United Kingdom, ab184787), and β-actin (dilution 1:3000; Proteintech, United States, 60008-1-Ig; Table ).

Techniques: Expressing, Western Blot, Comparison, Control

Journal: PLoS ONE

Article Title: HIV-1 Tat Interacts with and Regulates the Localization and Processing of Amyloid Precursor Protein

doi: 10.1371/journal.pone.0077972

Figure Lengend Snippet: ( A ) GST and GST-Tat were purified on glutathione-Sepharose beads. The beads were boiled to elute the bound proteins, which were then run on a 12% SDS-PAGE gel and stained with Coomassie brilliant blue (left panel). GST pulldown assay with SK-N-MC neuroblastoma cell lysates shows a strong interaction between APP and GST-Tat (right panel). SK-N-MC neuroblastoma cell extracts incubated with GST- or GST-Tat-coated beads for 3 hours. The beads were washed three times with PBS and the eluted proteins were analyzed by western blotting with an anti-APP antibody (22C11). ( B ) Coimmunoprecipitation of Tat and APP in HEK 293FT cells transfected with Tat and/or Myc-tagged APP695 vectors. Proteins were precipitated with anti-Tat or anti-APP (6E10) antibodies and immunoblotted with anti-Tat or anti-Myc antibodies. ( C ) Coimmunoprecipitation of U-87 MG cell lysates transduced with mock, Lenti-Tat, or Lenti-mTat virus. APP was precipitated with an APP antibody (6E10), and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (22C11) or anti-Tat antibodies. Reciprocally, Tat was precipitated with anti-Tat antibody, and the precipitate was analyzed by SDS-PAGE followed by Western blotting with anti-APP (A8717) or anti-Tat antibody. ( D ) Purified recombinant APP interacts with GST-Tat. Purified recombinant APP (500 ng) was incubated with GST- or GST-Tat-coated beads and the eluted proteins were analyzed by western blotting with an APP antibody. A large amount of recombinant APP bound to the GST-Tat beads. ( E ) Tat interacts strongly with APP. SK-N-MC neurobalstoma cell lysates were incubated with GST, GST-Tat, or GST-Tat beads, and washed three times in buffer containing 137, 200, 300, 400 or 500 mM NaCl. APP remained associated with GST-Tat under high-salt conditions. ( F ) The cysteine-rich domain of Tat is important for association with APP. Deletion mutants were produced as GST-fusion proteins and subjected to GST-pulldown assays with SK-N-MC cell lysates. L, load; B; bound.

Article Snippet: Purified recombinant APP (cat. no APP-526H, Creative BioMart, NY, USA) was resuspended in PBS supplemented with 1% NP-40 to yield a final concentration of 1 ng/μl, and 500 μl was incubated with GST- or GST-Tat-coated beads for 3 hours at 4°C.

Techniques: Purification, SDS Page, Staining, GST Pulldown Assay, Incubation, Western Blot, Transfection, Transduction, Virus, Recombinant, Produced