Figure 2.

Arginine topology controls cell binding and uptake. (A) Surface binding of rhodamine labeled cationic miniature proteins in the absence of endocytosis and removal by trypsin treatment. HeLa cells were treated with 1 μM rhodamine labeled cationic miniature protein for 30 min. Cell were then treated with trypsin (0.05%, 10 min, 37°C) or PBS before washing and analysis by flow cytometry. (B) Fraction of cell-associated fluorescence remaining after trypsin treatment. These data represent the ratio of black to red bars shown in A. (C) Cell uptake of rhodamine labeled peptides by HeLa cells after 30 min and 90 min.AlexaFluor-488-transferrin (Tf⁴⁸⁸) when added to HeLa cells (D) colocalizes with (E) Tf⁵⁴⁶ and (F) rhodamine labeled miniature proteins. Perfect colocalization is characterized by a Pearson's R value (R) equal to 1, while R values near 0 represent little or no colocalization. The correlation value observed when cells were treated with both Tf⁴⁸⁸ and alexa-fluor-546-labeled transferrin (Tf⁴⁶) is 0.905. (G) When added with Tf⁴⁸⁸, aPP^R shows little intracellular signal. Rhodamine labeled cationic miniature proteins and Tf⁵⁴⁶ are shown in red, Tf⁴⁸⁸ is shown in green, Hoescht (nucleus) is shown in blue. See also Figure S2.