Journal: RSC Chemical Biology

Article Title: Origin, toxicity and characteristics of two amyloid oligomer polymorphs

doi: 10.1039/d1cb00081k

Figure Lengend Snippet: Immuno-staining of early vs. late stage amyloid oligomers. Dot blots of different amyloid aggregates and controls at three different concentrations. (A) Reactivity of A11 with RFs (top row) and late-stage oligomers (middle row). (B) Ponceau staining of all samples blotted onto membrane. Concentrations for the various samples that yielded equivalent protein retention were (from left to right) RFs: 2.0, 1.0 and 0.5 μg; all others: 12, 8 and 4 μg.

Article Snippet: Membranes were blocked with 7% milk in 1× Tris-buffered saline (TBS) at room temperature for 1 h. After three washing steps in 1× TBST (5 min each), membranes were incubated overnight at 4 °C with 1 : 1000 dilution of the amyloid oligomer antibody A11 (Novus Biologicals).

Techniques: Immunostaining, Staining, Membrane

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