Figure 8. Calcium dependent internalization of HB-19 that binds the RGG domain of surface nucleolin.

HeLa cells were passaged in DMEM culture medium. Two days later fresh DMEM (in A and C) or RPMI (in B and D) culture medium was added to the cell monolayer in the absence or presence of 5 mM EGTA (B) or CaCl₂ (D) as indicated. Internalization of HB-19/Btn (5 µM) was then carried out at 37°C for 90 min before fixation with PFA-Triton and processing for confocal microscopy. Fixed cells were successively incubated with rabbit anti-biotin and goat Texas Red-conjugated anti-rabbit IgG. A scan corresponding to a cross-section toward the middle of the cell monolayer is shown.