Journal: NPJ Parkinson's Disease

Article Title: Glycation modulates glutamatergic signaling and exacerbates Parkinson’s disease-like phenotypes

doi: 10.1038/s41531-022-00314-x

Figure Lengend Snippet: Wild-type littermate (WT) and Thy1-aSyn transgenic mice received an intracerebroventricular (ICV) injection of MGO or vehicle (PBS) and protein brain extracts from several regions analyzed 5 weeks post injection. Protein extracts were resolved by SDS-PAGE or loaded into membranes in a dot-blot system (see Supplementary Fig. ). Membranes were probed with anti-aSyn, anti-CEL, anti-pS129-aSyn and anti-β-actin for normalization. Representative blots showing two samples from each experimental group are shown for aSyn, and densitometric analysis represented for aSyn and CEL in a , b midbrain, c , d striatum, e , f cerebellum, g , h prefrontal cortex, and i , j hippocampus, respectively. k Representative blot showing four samples from vehicle- and MGO-injected Thy1-aSyn mice are shown for pS129-aSyn and aSyn total signal. Densitometric analysis of the ratio between pS129-aSyn and aSyn is presented. l Representative blot showing aSyn probing in soluble and insoluble fraction from vehicle- and MGO-injected Thy1-aSyn mice. Densitometric analysis of the ratio between the insoluble and the sum of both soluble and insoluble signals is presented. At least n = 5 in all groups, data in all panels are average with error bars representing standard deviation, Ordinary one-way ANOVA, * p < 0.05, ** p < 0.01, **** p < 0.0001; unpaired two-tailed t -test with equal SD, # p < 0.05.

Article Snippet: Membranes were incubated with blocking solution (5% bovine serum albumin) in 1× TBS (20 mM Tris, 136 mM NaCl, pH 7.6) at room temperature for 30 min. Primary antibody incubations were carried out overnight at 4 °C, using given concentrations in blocking solution: CEL (Mouse Anti-N ε -carboxyethyl lysine, in a dilution of 1:1000 in blocking solution, Cosmo-Bio, USA), aSyn (Purified Mouse Anti-α-Synuclein antibody, in a dilution of 1:1000 in blocking solution, BD Biosciences; San Jose, CA, USA), phosphorylated aSyn at residue 129 (pS129, [J18] SMC-600, StressMarq; Victoria, Canada), glyoxalase I (Rabbit polyclonal anti-Glyoxalase I antibody – FL-184, sc67351, Santa Cruz Biotechnology), and β-actin (Mouse Monoclonal anti-β-actin antibody in a dilution of 1:5000 in blocking solution, Ambion, Thermo Fisher Scientific; Waltham, MA, USA).

Techniques: Transgenic Assay, Injection, SDS Page, Dot Blot, Standard Deviation, Two Tailed Test