Nov 01, 2024
MLSA5 and VPS34 inhibitor treatments and immunoblotting of whole-cell lysates from cell culture systems
- Felix Kraus1,
- Harper JW2,3
- 1Department of Cell Biology, Blavatnik Institute, Harvard Medical School, 240 Longwood Ave, Boston MA 02115, USA;
- 2Harvard Medical School;
- 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Felix Kraus, Harper JW 2024. MLSA5 and VPS34 inhibitor treatments and immunoblotting of whole-cell lysates from cell culture systems. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv526r5v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 01, 2024
Last Modified: November 01, 2024
Protocol Integer ID: 111353
Keywords: ASAPCRN, atg8 lipidation after casm induction, assessing atg8 lipidation, hela cell culture system, cell lysates from cell culture system, cell lysate, vps34 inhibitor treatment, cell culture system, immunoblotting, mlsa5, cell
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-025160
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Disclaimer
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Abstract
This is a protocol for assessing ATG8 lipidation after CASM induction by Western blotting from whole-cell lysates derived from HeLa cell culture systems.
Troubleshooting
Safety warnings
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Cell Culture and Treatments
5m
Seed HeLa TMEM192-3xHA cells of desired genotypes into 6-well plates.
Upon reading desired confluency (~70%), treat cells according to the following four treatments:
1. Fed
2. 3 h with the VPS34 inhibitor SAR405 (1 µM)
3. 2 h with MLSA5 (10 µM)
4. 2 h VPS34 inhibitor + MLSA5
Aspirate growth media, wash twice with 1xPBS and harvest cells in ice-cold PBS on ice by scraping cells from the wells.
SDS-PAGE and immunoblotting
5m
For whole-cell lysates, prepare samples following standard protocols, and final samples should be in LDS buffer with DTT or similar. Incubate samples at 80 °C for 00:05:00 .
5m
Load samples into a NuPAGE Novex‱ 4-12% Bis-Tris Midi Protein Gels and separate by electrophoresis in 1xTris/Glycine/SDS buffer.
Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol Tis/Glycine buffer.
Block membrane in blocking buffer (5% non-fat dry milk or 3% BSA in TBST) at Room temperature for 01:00:00 .
1h
Incubate membrane in primary antibody solution (blocking solution plus primary antibody at 1:500-1:1,000, depending on the primary antibody) at 4 °C for 12:00:00 to16:00:00 .
Primary antibodies:
LC3B
panGARBARAP
Actin
1d 4h
Wash membrane six times with TBST for 00:05:00 each wash.
5m
Incubate membrane in secondary antibody solution (blocking solution plus secondary antibody conjugated to HRP at 1:5,000-1:10,000) at Room temperature for 01:00:00 .
1h
Wash membrane four times with TBST for 00:05:00 each wash.
5m
Apply Western Lightning Plus Chemiluminescence substrate (Revvity) to membrane and acquire blot images using a ChemiDoc MP imager.
Process raw image files with Image Lab software (Bio-Rad).