Chemiblot xrs system

Quantified proteins were first denaturalized by adding an equal volume of Laemmli buffer (Bio-Rad) supplemented with β-mercaptoethanol (Sigma Aldrich). After that, proteins were separated by SDS-PAGE using 4% pre-cast acrylamide gels (Bio-Rad) in TGS buffer (Bio-Rad). After electrophoresis, proteins were transferred from the gel to a nitrocellulose membrane (Bio-Rad) using a TransBlot turbo system from Bio-Rad. Membranes were then blocked for 1 h with 3% skim milk (Sigma Aldrich) in PBS. Membranes were subsequently washed PBS-Tw prior to overnight incubation with primary antibodies (Table 2). After several washes with PBS-Tw, the membranes were incubated with peroxidase-conjugated secondary antibody for 2 h at 1:10,000 dilution. After final washes with PBS-Tw, blots were developed using ECL Clarity and ChemiBlot XRS + system from Bio-Rad. β-actin was used as a housekeeping protein, and data are expressed as a percentage of the fold to actin.Table 2

Antibodies used for western blot

Antibody	Company	Headquarters	Reference	Dilution
Rabbit anti-hSYN	Abcam	Cambridge, UK	AB138501	1:1000/1:5000
Rabbit anti-pSYN P129	Abcam	Cambridge, UK	AB168381	1:3000
Goat anti-GAL3	R&D Systems, Bio-techne	Minneapolis, MN, USA	AF-1197	1:1000
Mouse 5C2 anti-αSyn	Enzo Life Sciences	Farmingdale,NY,USA	ALX-804-656-R100	1:1000
Mouse anti-β-ACTIN-Peroxidase	Sigma Aldrich	St Louis, MO, USA	A3854	1:10,000

Western blotting was used to measure APP levels in brain extractions. The proteins were extracted from the cortex using PBS or RIPA buffer (Sigma-Aldrich, Germany) with proteinase and phosphatase inhibitors. The blots were run using pre-casted gels (4–20% from Bio-Rad) in TGS buffer (Bio-Rad). The proteins were transferred to nitrocellulose membranes (Bio-Rad) using the TransBlot turbo system from BioRad. The membranes were blocked for 1h with skim milk at 3% in PBS, then washed 3 × 10 mins in PBS and Tween-20 at 0,1%, after which they were incubated with primary and secondary antibodies diluted in PBS-Tween 20 at 0.1%. To develop the blot we use ECL Clarity (Bio-Rad) according to the manufacturer’s protocol and ChemiBlot XRS + system from Bio-Rad.

Proteins were extracted from cell cultures with RIPA buffer (Sigma-Aldrich, Germany) along with proteinase and phosphatase inhibitors (Roche, Switzerland). Protein concentration was measured using a BCA kit according to the manufacturer’s protocol (BCA Protein Assay-Kit, ThermoScientific, Sweden). Protein extracts were then separated by SDS-PAGE using pre-cast gels (4–20%, Bio-Rad) in TGS buffer (Bio-Rad, Sweden). The proteins were transferred to nitrocellulose membranes (Bio-Rad, Sweden) using the TransBlot Turbo system from Bio-Rad. The membranes were subsequently blocked for 1 h with skim milk at 3% (w/v) in PBS, then washed 3 × 10 min in PBS supplemented with 0.1% (w/v) Tween 20 (PBS-T). Then, blots were incubated with primary antibodies in PBS-T, as mentioned above, overnight. Following this, we incubated the blots with secondary antibodies for 2 h. After the secondary antibody, we washed three times with PBS-T, and then the blots were developed using ECL Clarity (Bio-Rad) according to the manufacturer’s protocol and imaged using the ChemiBlot XRS+ system from Bio-Rad.