0.2 m pvdf membrane

Retinal tissues (N = 3 biological replicates) were lysed with RIPA buffer (Thermo Fisher Scientific, Cat.# 89900) containing protease and phosphatase inhibitors (Thermo Fisher Scientific, Cat.# A32961), and protein concentrations were measured using the DC Assay (Bio-Rad Laboratories, Cat.# 5000114), according to the manufacturer’s protocol. An equal amount of protein samples was loaded and separated on an SDS-PAGE 7.5% PROTEAN TGX Stain-Free gel (Bio-Rad, Cat.# 4568024), then transferred to 0.2 µm PVDF membranes (Bio-Rad Laboratories, Cat.# 1,704,156). The PVDF membranes were blocked in 5% Nonfat dry milk (Bio-Rad Laboratories, Cat.# 1,706,404) in Tris-buffered saline with 0.1% Tween 20 (TBST) (VWR, Cat.# K873-4L). Samples were probed with the primary antibodies listed in Additional file 1: Table S1 overnight in 5% bovine serum albumin (Gold Biotechnology, Cat.# A-420-100) in TBST. Blots were incubated with horseradish peroxidase-conjugated species-specific secondary antibodies listed in Additional file 1: Table S1. Proteins were visualized with an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Cat.# 34095) and digitally imaged on a ChemiDoc MP Imaging System (Bio-Rad Laboratories). Quantification of the band intensity was carried out using the Image J Software (NIH, Bethesda, MD).

Conditioned medium from three independent differentiations of two control and two MSA-P (P1, P3) iPSC cell lines was collected, each at 40 and 70 ± 4 days of differentiation into MSNs. To remove cell debris, samples were spun at 1000× g for 10 min. Using vacuum dot blot equipment, the proteins were transferred on 0.2 µm PVDF membranes (Bio-Rad Laboratories). After blocking the membranes for 2 h at room temperature with 30% RotiBlock solution (Carl Roth, Karlsruhe, Germany), primary antibodies (Supplementary Table S1) were diluted accordingly and incubated with the membranes overnight at 4 °C. Membranes were washed (Tris-buffered saline (TBS) supplemented with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA)), and the appropriate HRP-coupled secondary antibodies (Supplementary Table S2) were incubated for 1 h at room temperature. For visualization, the membranes were incubated in Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA), and the images were acquired using the Odyssey Fc (LICOR Biotechnology, Lincoln, NE) imaging system. Image Studio™ Software (version 5.2.5, LI-COR Biosciences GmbH, Bad Homburg vor der Höhe, Germany) was used for signal intensity annotation, and α-synuclein signals were normalized against GAPDH and β-actin signals.

PBMCs were rinsed twice with PBS and collected by centrifugation, then lysed on ice in RIPA lysis buffer supplemented with protease inhibitor cocktail (Beyotime). After determining the protein concentration using the BCA protein assay kit (Beyotime), the appropriate sample solution volumes were calculated and mixed with the loading buffer (Bio-Rad). Proteins were separated via electrophoresis using 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred to 0.2-µm PVDF membranes (Bio-Rad). The membranes were incubated overnight at 4°C with the following primary antibodies: rabbit antiACTB (1:50,000, AC026, ABclonal) and mouse antiGAPDH (1:20,000, 60004-1-Ig, Proteintech). Afterward, corresponding secondary antibodies including IRDye 680RD goat anti-mouse and IRDye 800CW goat anti-rabbit (1:15,000, Li-Cor) were incubated for 1 h at room temperature. Protein blots were detected using the Odyssey CLx system (Li-Cor).

Cells or tissues were homogenized in lysis buffer (10 mM Tris–HCl, pH 7.6; 1% Triton X-100; 1 mM EDTA; 0.1% sodium deoxycholate) supplemented with protease and phosphatase inhibitors (Complete and PhosSTOP, Roche, Basel, Switzerland). Protein concentrations were determined by DC-Protein Assay (Bio-Rad, Hercules, CA, USA). Equal amounts were separated on SDS–polyacrylamide gels and transferred to 0.2 µm- PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked, incubated with primary antibodies and secondary antibodies (Merck-Millipore, Billerica, MA, USA or Dako, Glostrup, Denmark) afterwards. Primary antibodies used were against AKT, P-AKT (Ser473), GSK3β, P-GSK3β (Ser9), FAK, P-FAK (Tyr397), HSL, P-HSL (Ser660) (Cell Signaling Technology, Danvers, MA, USA), INTB1, P-INTB1 (Thr788/9) (Abcam, Cambridge, United Kingdom), Tubulin, Actin or GAPDH (Sigma-Aldrich, St. Louis, MO, USA). Immunoblots were detected by chemiluminescence (Pierce ECL Western Blotting Substrate, Thermo Fisher Scientific Waltham, MA, USA) and imaged with ImageQuant LAS 500 System (General Electric Healthcare, Little Chalfont, United Kingdom). Densitometries were measured using ImageJ software (NIH).

A defined amount of 20 µg per lane of protein was subjected to 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue. Unstained replicas were transferred to 0.2 µm PVDF membranes (Bio-Rad, 170-4156) using a Bio-Rad Trans-Blot Turbo TM (0.6 Å, 25 V, 40 min). Western blot analyses were carried out using sera obtained from mice immunized with PP2A [9 (link)] and diluted 1 : 150 with PBS-T (0.3% tween-20 in PBS). Membranes were washed with phosphate-buffered saline with 0.05% Tween 20 (PBS-T), incubated for 2 h at 37°C with 1 : 1000 polyclonal Goat anti-Mouse Immunoglobulins/HRP (Dako Cytomation-P0447) and developed using chemiluminescence (ClarityTM Western ECL Substrate, Bio-Rad, 170-5060).
The protein band from the SDS-PAGE electrophoresis recognized by the sera was sequenced by fingerprinting and identified at the Servicio de Proteómica del Centro de Biología Molecular Severo Ochoa (CBMSO) in Madrid (Spain) in an Autoflex matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker) equipped with a reflector following a previously described protocol [75 (link)].

RIPA buffer (Thermo Scientific) containing Complete EDTA-free protease inhibitor was used to isolate proteins from cells and tissues. Proteins (30 μg) were loaded with a 4–15% TGX gel (BioRad) and then transferred to 0.2 µm PVDF membranes (BioRad). The membranes were blocked in 5% non-fat dried milk/Tris-buffered saline and 0.1% Tween-20 for 1 h at room temperature. Next, the membranes were incubated with primary antibodies: anti-SIPL1 (Abcam, ab79039), anti-MAZ (Abcam, ab85725), anti-AKT (Abcam, ab18785), anti-p-AKT (Abcam, ab38449), anti-P65 (Abcam, ab32536) or anti-p-P65 (Abcam, ab76302), and anti-GAPDH (Abcam, ab9485) overnight, along with horseradish peroxidase-labeled IgG (Abcam, ab205718) at room temperature. The protein bands were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Densitometry analysis was performed using ImageJ software (version 1.36; National Institutes of Health, Bethesda, MD, USA).

Cells were seeded at density 3 × 10⁵ in a six-well plate for 24 h. The cells were treated with PE5 at concentrations 0–100 µM for 24 h (concentration dependent) and were treated with PE5 at 50 µM for 6, 12, 24, and 48 h (time dependent). Then, cells were incubated on ice for 40 min with RIPA buffer, 1% Triton X-100, 100 mM PMSF, and a protease inhibitor. Cell lysates were analyzed for protein content using the BCA protein assay kit from Pierce Biotechnology (Rockford, IL, USA). Equal amounts of denatured protein samples (60 µg) were loaded onto 10–15% SDS-PAGE for various proteins. Then, the gels were transferred to 0.2 µm PVDF membranes (Bio-Rad, Hercules, CA, USA). The transferred membranes were blocked with medium (25 mM Tris-HCl (pH 7.5), 125 mM NaCl, and 0.05% Tween20 (TBST)) containing 5% non-fat dry milk powder for 1 h, and were incubated overnight with specific primary antibodies. After the overnight block, the membranes were washed three times with TBST and were incubated with the following appropriate horseradish peroxidase-labelled secondary antibodies: anti-rabbit IgG or anti-mouse, for 2 h at room temperature. The immune complexes were detected by SuperSignalWest Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA) and were exposed to the film.