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Data represent means ± SD of n ≥ 3 separate determinations PSMA-specific ligand internalization was determined by incubation of LNCaP cells (37 °C, 60 min) with the respective radioligands (0.2 nM) in the absence (total internalization) and presence (non-specific internalization) of 10 μM PMPA.
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In addition, the dose response curve of EGFR-NCE was the only one displaying a threshold-controlled behaviour, since both EGFR-CME and EGFR-total internalization showed completely different behaviours that did not match that of EGFR ubiquitination.
In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy).
β1AR internalization was determined by total internal reflection fluorescence (TIRF) in cells stably expressing C-terminally YFP-fused β1AR.
Data for total internalized ligand (mean ± SD) were corrected by non-specific internalization (mean ± SD) at the respective time point using Excel considering the laws of error propagation.
To determine particle internalization, total particles and internalized particles were determined visually and the data is presented as percent of cell associated particles that are internalized.
The lowest dose (1 nM) of the ligand supports endocytosis for up to 20 min, achieving the highest total level of internalization at ∼50%.
Internalization experiments were also performed at 4°C demonstrating a reduction of both total and internalized radioactivity (p<0.05).
Finally, in the revised manuscript, we chose not to show calculated internalization efficiencies (internalized CTB divided by total CTB bound), out of concern that the low amount of total CTB bound under some conditions could comprise the accuracy of these calculations.
(A) Internalized fraction calculated versus cell-associated activity and (B) internalized fraction calculated per million cells versus total added radioactivity; nonspecific internalization, as determined in the presence of 1 μM Tate in the medium, is indicated by dashed lines.
The comparison of 124I-J591 and 89Zr-J591 yielded similar model-derived values of the total antigen concentration and internalization rate.
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