TY  - JOUR
TI  - Monitoring Endocytosis of Integral Membrane Proteins Using Western Blot-Based Detection of Biotinylated Antibody Uptake
AU  - Graninger, Alexandra 
AU  - Satpute-Krishnan, Prasanna 
VL  - 15
IS  - 22
PY  - 2025
DA  - 2025/11/20
SP  - e5511
C1  - Bio-protocol 2025;15:e5511
DO  - 10.21769/BioProtoc.5511
UR  - https://doi.org/10.21769/BioProtoc.5511
AB  - The antibody-uptake assay is a commonly used technique to monitor endocytosis of integral membrane proteins including transmembrane and glycosylphosphatidylinositol-anchored proteins (GPI-APs). The antibody-uptake assay typically involves incubating live cells with fluorophore-conjugated antibodies directed against the extracellular domain of the integral membrane protein of interest. Antibody uptake is then detected by flow cytometry or confocal microscopy. However, these detection modalities may be inaccessible to some labs or require extensive training to operate. Thus, we developed an easy and novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot-based approach to the antibody-uptake assay that exploits the strong affinity between biotin and streptavidin. Instead of incubating cells with fluorophore-conjugated antibodies to monitor antibody uptake, our assay involves incubating cells with biotinylated antibodies, processing the cell lysates for western blot, and probing the membrane with detectably conjugated streptavidin. From preparation to quantification, this protocol requires less hands-on time than other approaches and is amenable to small-scale drug or siRNA screens. Here, we demonstrate the utility of our approach using the well-characterized misfolded GPI-AP, YFP-tagged C179A mutant of prion protein (YFP-PrP*), as our model substrate. YFP-PrP* constitutively traffics to the plasma membrane (PM), where it binds to anti-GFP antibody, and immediately undergoes endocytosis to lysosomes. To validate our protocol, we present measurements of antibody uptake under conditions known to enhance or inhibit YFP-PrP*’s traffic to the PM. Using this assay, we present new evidence that, under certain conditions, YFP-PrP* is able to undergo degradation via a pathway that does not involve exposure on the cell surface.
KW  - Antibody uptake
KW  - Integral membrane protein trafficking
KW  - Plasma membrane
KW  - RESET
KW  - Internalization
KW  - Transmembrane and GPI-anchored protein
KW  - Secretory pathway
KW  - Endocytosis
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False