Hutu80 and NCI h716 treatment with SCFAs and α synuclein Western Blotting

Hutu80 and NCI h716 parental cells were seeded in wells at a density of 1.0 10⁵ cells the day before treatments and incubated at 37°C in a 5% CO₂ atmosphere

Cells were treated with differents concentrations ( 0.5 ; 1 ; 2 ; 4 and 6 mM) of SCFAs Acetate, propionate and butyrate (100 mM stock solutions in water (SIGMA-ALDRICH)) and with or not CBE 10 uM (Cyaman chemical) for differents time of incubation ( 2h ; 6h ; 24h and 48h). For CBE treaments, cells were pre-incubated with 10 uM CBE for 4h 0m 0s and then incubated with SCFAs.

Cells were lysed in lysis buffer containing protease inhibitors (see materials)

-For HuTu80 cells lysis (Adherent cells):

Carefully remove culture medium and wash cells 2 times with cold PBS. Add 100 uL of cold lysis buffer to the cells. Keep on ice for 0h 5m 0s , stirring the plate occasionally for uniform spreading.

Collect the lysate on one side using a cell scraper, collect the lysate and transfer it to a microcentrifuge tube. Centrifuge samples at 14000x g,4°C,0h 0m 0s for 0h 15m 0s to collect cell debris.

Transfer the supernatant to a new tube and store at -20°C until further use.

• For NCI h716 cells (Non-adherent cells) :

Collect cells by centrifugation at 2500x g,0h 0m 0s for 0h 5m 0s .

Discard supernatant and wash cells twice in cold PBS. Collect cells by centrifugation at 2500x g,0h 0m 0s for 0h 5m 0s. Add 100 uL lysis buffer to the cell pellet and stir up and down to suspend the pellet.

Centrifuge the mixture at 14000x g,4°C,0h 0m 0s for 0h 15m 0s to sediment cell debris.

Transfer the supernatant to a new tube and store at -20°C until further use.

Note: To increase yields, sonicate the pellet for 30 seconds with a 50% pulse before centrifuging.

Before immunoblotting, quantities of extracted protein were determined using BCA assays (Pierce^TM BCA Protein Assay Kit)

Samples preparation : Mix samples with Laemelli buffer (see materials) containing 100 mM DTT and heat the samples 95°C 0h 10m 0s

Samples preparation : Centrifuge samples 0h 0m 30s to collect condensation from tube lid and Vortex samples 0h 0m 10s

Electrophoresis and transfer :

Separate samples (~ 10 ug total protein) by polyacrylamide gel electrophoresis using precast TGX Stain-free pre-cast SDS-polyacrylamide gel (Bio-rad)and migration buffer (see materials) at 200 V 0h 30m 0s or until dye front reaches the bottom of the gel. Run with pre-stained size markers

Proteins were then transferred using liquid transfer on PVDF membranes (Bio-rad). PVDF membrane should be imbibed in EtOH 100% and equilibrate with transfer buffer (see materials) before use.

Soak transfer sandwich components (4 sheets of filter paper and 5 blotting pads) in transfer buffer and assemble in the transfer cassette in the following order:

Starting with the cathode plate

1 x blotting pads

2 x filter paper

gel

PVDF

2 x filter paper

1 x pad

Use a rollerin every step in order to eliminate air bubbles

Place cassette and a cooling unit in transfer tank placed in glace and transfer protein 100V 1h 0m 0s in transfer buffer

Immunodetection:

Incubate membrane in 4% paraformaldehyde in order to fix a synuclein protein

Wash membrane with TBST (see materials) 4 times 0h 5m 0s

Block non-specific binding sites in the membrane with block solution (TBST 5% skimmed milk) for 1h 0m 0s

Incubate membranes in block solution containing rabbit monoclonal antibody against α-synuclein (1:200) (ab212184) and β-actin (1:10000) 4°C

Wash with TBST 3 x0h 5m 0s

Incubate membrane with secondary antibody for 1h 0m 0s atRoom temperature

Wash with TBST 3 x0h 5m 0s

For chemiluminescent detection use an Bio-rad ECL plus kit according to the manufacturer's instructions : Add 1 mL of ECL per membrane and incubated for 0h 5m 0s

Visualise detected protein using iBright imaging system (Thermofisher)

After visualisation membranes can be washed with TBST 3x0h 5m 0s and conserved at 4°C if it will be reused.