Figure 2.

Zfra or WWOX peptide upregulates the binding of endogenous WWOX with specific proteins in organs of mice, which correlates with cancer growth suppression. (A) BALB/c mice received tail vein injections of Zfra4-10 and/or WWOX7-21 peptides, followed by inoculation with 4T1 breast cancer cells two weeks later and sacrifice 18 days later. (B) By co-immunoprecipitation, binding of HIF-1α with C1qBP and COX2 was increased by 30 to 50% in mice treated with Zfra4-10. Additionally, increased binding of WWOX with C1qBP and other indicated proteins was shown in the spleen of mice treated with either Zfra4-10 or WWOX7-21 peptide (>50 to 90%). These events correlate with cancer growth suppression. In combination of both peptides, the complex formation of WWOX and target proteins was reduced, which correlates with increased tumor growth. The Whole Blots for Western Blot analysis for Figure 2B are shown in Figure S8. (C) Endogenous WWOX strongly bound to Iba1, Oct4, ERK, NF-κB p65, and GFAP in the lung of mice treated with Zfra4-10 or WWOX7-21 peptide. In controls, non-immune serum was used for co-immunoprecipitation and equal loadings of protein A/G beads are reflected by IgH bands. The Whole Blots for Western Blot analysis for Figure 2C are shown in Figure S9. (D) Binding of WWOX with presenilin-1 was not observed in the brain of mice treated with PBS, or Zfra4-10 and/or WWOX7-21 peptides. (E) The binding status of pY33-WWOX with NF-κB p65 is shown in the kidney. The Whole Blots for Western Blot analysis for Figure 2D,E are shown in Figure S10. Figures with digital data for Western blots 2B to 2E are shown in Figure S12.