Figure 2

rVAR2 internalization depends on binding to cell surface CSA. (a) Immunofluorescence of rVAR2 in COLO205 and PC3 cells after 2 h incubation. (b) quantification for mean pixel intensity of AlexFluor488 signals in rContr, rVAR2, and rVAR2+ CS treated cells. Statistical analysis was performed on n ≥ 30 cells, ****p ≤ 0.0001 by ANOVA followed by Tukey’s post hoc analysis. Scale bar represents 10 µm. (c) Western blot for intracellular rVAR2 follow rVAR2 treatment in the presence or absence of CSA competition. (d) Flow cytometry analysis of rVAR2 binding in COLO205 and PC3 cells with and without SC treatment. (e) Western blot of internalized rVAR2 in COLO205 and PC3 cells after SC treatment at the indicated concentrations. rVAR2 was incubated for 1 h and vinculin was used as loading control. (f) Immunofluorescence images of rVAR2 after 1 h incubation in COLO205 and PC3 cells following SC treatment. Scale bar represents 10 µM. (g) CHST11 mRNA levels in PC3 and COLO205 cells as determined by qPCR and normalized to GAPDH. (h) rVAR2 binding analysis 48 h following control and CHST11 siRNA transfections in PC3 and COLO205 cells. (i) Western blot of internalized rVAR2 in PC3 and COLO205 cells following CHST11 knockdown, vinculin was used as loading control.