Nucleolin Binding Assay Protocol

The nucleolin binding assay was performed as previously described with minor modifications [14 (link),24 (link)]. Briefly, 2 mM EDTA was used to detach the cells and they were put on ice to cool. All of the treatments occurred at 4 °C to ensure that no cellular uptake could occur. The cells were treated with RNA or DNA oligonucleotides (1 µM) for 15 min prior to incubation with an anti-nucleolin antibody (Ab22758, Abcam, Cambridge, UK) for 30 min at 4 °C, at a concentration of 1 µg. Subsequently, the cells were stained with a secondary Alexa 488-conjugated donkey anti-rabbit IgG secondary antibody (5 µg/mL; Invitrogen, Massachusetts, MA, USA) for 30 min at 4 °C, followed by the acquisition of the samples using a FACSVerse cytometer (BD, New Jersey, NJ, USA). The analysis was performed using FlowJo software (Tree Star). Alternatively, after the secondary antibody staining, the cells were placed in imaging dishes and analyzed on a Zeiss Axio Observer Z1 microscope using a 20× objective followed by analysis using Zen Blue 3.2.