TITLE: Assessing autophagy using the HaloTag-LC3B cleavage assay
AUTHORS: Marvin Skulsuppaisarn

[DESCRIPTION]
Assay to detect HaloTag-LC3B during starvation autophagy based on  https://doi.org/10.7554/eLife.78923.

[STEPS]
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
1. Generate HeLa cells expressing HaloTag-LC3B using pMRX-IP-HaloTag7-LC3 from Mizushima lab (Addgene #184899; DOI: 10.7554/eLife.78923).
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
2. Seed 400-450K cells in a six-well plate one day prior to the experiment.
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
3. Feed cells with 1 mLof complete DMEM for 60 min 

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
4. Replace with 1 mLof complete DMEM containing 50 nanomolar (nM) TMR-conjugated Halo ligand (Promega, GA1120) and incubate at 37 °C  for 20 min

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
5. Wash cells thrice with 1x PBS followed by incubation in 2 mL of EBSS (Gibco, 24010043) for 360 min

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6. Following starvation, harvest cells via cell scraping on ice .

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.1. Aspirate off DMEM and wash cells in 1 mL  of ice cold 1x PBS.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.2. Scrape cells from each well in 600 µL  of ice-cold 1x PBS and transfer into chilled 1.5 ml Eppendorf tube.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.3. To collect residual cells, add another 600 µL  of ice-cold 1x PBS transfer into 1.5ml eppendorf tube.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.4. Spin down cells at 10000 x g, 4 °C  for 1 min and aspirate off PBS.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.5. Perform a quick-spin at 10000 x g, 4 °C  and aspirate off residual PBS.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
6.6. Cell pellets can be frozen at -20 °C  or immediately used for immunoblotting

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
7. Lyse cells in 1 xLDS NuPAGE‱ LDS Sample Buffer (Invitrogen, NP0007) containing 100 millimolar (mM)  DTT and Boil at 99 °C  on thermomixer with shaking.

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
8. Measure protein concentration using A280 setting on Nanodrop. Ensure sample concentration is between 4-6 mg/ml.
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
9. Load 20 ug of protein on 4-12% Bis-Tris gels (Invitrogen, WG1402A). Run gel using 3-step ramp up setting (100v for 10mins, 150v for 10mins and 180v for 55mins).


SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
10. Transfer protein using wet transfer method onto an Immobilon-P PVDF Membrane ( 0.45 µm pore size) using a Criterion blotter (Bio-rad, 1704070), 100v for 60 min.



SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
11. Wash and block PVDF membrane:
- 1x in PVDF destain for 1 min
- 3x in TBS-T (1xTBS/0.05% Tween-20) for 2 min each.
- Block in 10% Milk/TBS-T for 20 min
- 3x in TBS-T for 2 mineach
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
11.1. Cut the blots at the 65kDa molecular weight marker and incubate in primary antibodies made up in TBS-T/3% BSA overnight at 4 °C  on rocking platform
- >65kDa section in VCP (Cell signalling, 2648S) (1/1000 dilution)
- <65kDa section in anti-HaloTag (Promega, G9211) (1/100 dilution)
SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
12. Following overnight incubation, recycle antibodies back into tubes and wash PVDF membrane.
- 3x in TBS-T for 2 min each

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
13. Incubate in anti-mouse HRP (Cell signalling, 7076S) and anti-rabbit HRP secondary antibodies (Cell Signalling, 7074S) for 60 min
- HaloTag (1/10,000 dilution for secondary)
- VCP (1/5,000 dilution for secondary)

SECTION: Assessing starvation autophagy using the HaloTag-LC3B cleavage assay
14. Wash blots prior to developing in ECL prime (Cytiva, RPN2232SK)
- 2x in TBS-T for 2 min each.
- 2x in TBS for 2 min each.