Oct 31, 2024
NPC1 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. NPC1 inhibitor treatments and immunoblotting of whole-cell lysates from cell culture systems. protocols.io https://dx.doi.org/10.17504/protocols.io.261ger657l47/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: October 31, 2024
Last Modified: October 31, 2024
Protocol Integer ID: 111354
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-025160
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Disclaimer
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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.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.
Abstract
This is a protocol for assessing abundance of autophagy proteins after inhibition of the lysosomal cholesterol transprorter NPC1 via the U18666A inhibitor by Western blotting from whole-cell lysates derived from HeLa cell culture systems.
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
Cell Culture and Treatments
5m
5m
Seed HeLa TMEM192-3xHA cells of desired genotypes into 6-well plates.
Upon reading desired confluency (~50 - 70%, depending on the duration of inhibitor treatment), treat cells according to the following four treatments:
1. Fed
2. NPC1 inhibitor U18666A (2µM)
Treat cells for 1 to 3 days (depending on the experiment).
Change media and inhibitor daily.
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
SDS-PAGE and immunoblotting
5m
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:
NCOA4
FTH1
SQSTM1 / p62
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).