Figure 2: TIRFM analysis of fluorescent E6BSA-FITC molecules. (A) Brightfield (BF) and TIRFM images of steroid-starved N-38 cultures treated for 30 min with E6BSA-FITC, ICI 182,780 and E6BSA-FITC, or BSA-FITC. Dashed lines outline the cell body (CB) perimeter; puncta found out-side the perimeter (arrow) were not computed. Images show E6BSA-FITC binds to membranes of specific cells, and is inhibited by ICI 182,780. (B) Particle analysis (bar graph) of fluorescent puncta from TIRFM images revealed a significant number of fluorescent puncta present per cell for E6BSA-FITC treated cells compared to BSA-FITC treated cells. On average 5.875 ± 0.972 (n = 8 cells) fluorescent puncta were present in E6BSA-FITC treated cells compared to only 0.556 ± 0.242/cell for BSA-FITC (1 μg/ml; n = 9 cells). Pretreatment for 1 h with 1 μM ICI 182,780 reduced E6BSA-FITC binding (2.125 ± 0.295; n = 8 cells) compared to E6BSA-FITC. The presence of E6BSA-FITC on the plasma membrane was transient; 60 min after the removal of E6BSA-FITC only 3.2 ± 0.327 (n = 10 cells) fluorescent puncta were present (TIRFM image not shown). (C) Schematic of E6BSA-FITC internalization and trafficking at the plasma membrane of N-38 neurons (Bonferroni post hoc test, **p < 0.001, ***p < 0.0001).