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. 2021 Jan 1;11(3):1326–1344. doi: 10.7150/thno.50564

Figure 3.

Figure 3

Cellular binding of NLN and NEW is mediated by CD44v6. (A) Western blotting analysis of CD44v6 and CD44 levels in MDA-MB231 cells after the knockdown (KD) of CD44v6 gene expression for 24, 48, and 72 h in cells treated with CD44v6 siRNA or control siRNA. GAPDH is used as a control protein. (B) The MFI of wild-type (WT) and CD44v6 KD MDA-MB231 cells bound to FITC-labeled NLN and NEW (25 µM). Data are shown as mean MFIs ± S.E. of peptide-bound cells from three separate experiments. *, P < 0.05 by Student's t-test. (C) Confocal microscopic images of WT and KD MDA-MB231 cells bound with FITC-labeled NLN and NEW (green, 25 µM) and stained with an anti-CD44v6 antibody (red) and DAPI (blue). Scale bars = 20 µm. (D) Competitive binding of FITC-labeled NLN and NEW (10 µM) following pre-treatment with anti-CD44v6 and anti-CD44 antibodies and IgG control in MDA-MB231 cells. The mean MFIs ± S.E. of peptide-bound cells from three separate experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA. (E) Competitive binding of an anti-CD44v6 antibody following pre-treatment with NLN and NEW (50 µM) in MDA-MB231 cells. ***, P < 0.001; n.s, not significant compared with untreated control by one-way ANOVA. (F) Pull-down assay of CD44v6 using biotin-labeled NLN and NEW and streptavidin beads, followed by immunoblotting with anti-CD44v6, anti-c-Met, and anti-CD44 antibodies. (G) Pull-down assays of the recombinant human CD44v6-Fc protein using biotin-labeled NLN and NEW and streptavidin followed by immunoblotting (IB) with anti-CD44v6 antibody. Ctrl, control peptide.