TITLE: Hutu80 and NCI h716 treatment with SCFAs and α synuclein Western Blotting
AUTHORS: Chaima Ezzine

[DESCRIPTION]
This protocol describe the treatment of human enteroendocrine cell models Hutu80 and NCI h716 parental cells with SCFAs and GCase inhibitor (CBE) in order to detect the impact of SCFAs and GCAse inhibition on alpha synuclein protein expression by immunoblotting.

[STEPS]
1. 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

2. 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 240 min  and then incubated with SCFAs.

3. Cells were lysed in lysis buffer containing protease inhibitors (see materials) 
3.1. -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 5 min , 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 14000 x g, 4 °C  for 15 min  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 2500 x g for 5 min . 
Discard supernatant and wash cells twice in cold PBS. Collect cells by centrifugation at  2500 x g for  5 min. Add 100 uL lysis buffer to the cell pellet and stir up and down to suspend the pellet.
Centrifuge the mixture at  14000 x g, 4 °C  for 15 min  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 (PierceTM BCA Protein Assay Kit)

4. Samples preparation : Mix samples with Laemelli buffer (see materials) containing 100 mM DTT and heat the samples 95 °C 10 min 

5. Samples preparation : Centrifuge samples 30 s    to collect condensation from tube lid and Vortex samples  10 s 

6. 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 30 min    or until dye front reaches the bottom of the gel. Run with pre-stained size markers

7. 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.
8. 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
9. Place cassette and a cooling unit in transfer tank placed in glace and transfer protein 100V 60 min  in transfer buffer

10. Immunodetection: 

Incubate membrane in 4% paraformaldehyde in order to fix a synuclein protein
11. Wash membrane with TBST (see materials) 4 times 5 min 

12. Block non-specific binding sites in the membrane with block solution (TBST 5% skimmed milk) for 60 min   

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

14. Wash with TBST 3 x5 min 

15. Incubate membrane with secondary antibody for 60 min atRoom temperature 

16. Wash with TBST 3 x5 min 
17. 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 5 min 
Visualise detected protein using iBright imaging system (Thermofisher)

18. After visualisation membranes can be washed with TBST 3x5 min  and conserved at 4 °C if it will be reused.