Analysis of Receptor Tyrosine Kinase Internalization Using Flow Cytometry

Abstract

SummaryThe internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

The 37kDa/67kDa Laminin Receptor acts as a receptor for Aβ42 internalization

“…It is noteworthy to add that very high cell densities negatively affect ligand binding efficiencies and thus 70% densities were considered optimal for successful internalization. Furthermore, cell signalling and receptor-mediated internalization events were synchronized as a result of serum starvation prior to experimentation30.…”

“…6b)29. All relevant controls were similarly incubated at 4°C, prior and post exogenously 500 nM Aβ 42 administration as this halts receptor-mediated internalization processes30. The cell surface levels of Aβ 42 in the untreated no internalization control (1 h, 4°C) was set to 100% in both experimental sets (Fig.…”

Characterization by flow cytometry of fluorescent, selective agonist probes of the A3 adenosine receptor

“…Cells were then incubated for up to 2 h at 4 or 37 °C. To study receptor internalization, after washing, cells were washed with 1 ml of ice-cold acid stripping buffer (DMEM with 0.2% BSA - bovine serum albumin - adjusted to pH 3.5 with HCl) three times for 5 min on shaking platform at 4 °C to remove surface bound ligand, and washed three times with ice-cold PBS for 5 min on shaking platform [23]. …”

Down-regulation of CMTM8 Induces Epithelial-to-Mesenchymal Transition-like Changes via c-MET/Extracellular Signal-regulated Kinase (ERK) Signaling

“…Flow Cytometric Analysis-Analysis was completed with a FACSCalibur (BD Biosciences) as described previously (33). Briefly, cells were detached with 5 mM EDTA and washed twice with ice-cold FACS buffer (PBS containing 0.2% BSA).…”