Figure 4: SARI inhibits VEGF expression by directly binding to Cp. | Nature Communications

Figure 4: SARI inhibits VEGF expression by directly binding to Cp.

From: RETRACTED ARTICLE: SARI inhibits angiogenesis and tumour growth of human colon cancer through directly targeting ceruloplasmin

Figure 4

(a) Co-immunoprecipitation products after adding anti-SARI antibody were separated by SDS–PAGE, and the differential band was analysed by MS. (b) Co-immunoprecipitation analysis of SW480-control and SW480-SARI cell lysates using anti-SARI and anti-Cp antibody. (c) Staining for Cp (green) and SARI (red) in SW480-control and SW480-SARI cells; DAPI staining for the nucleus. Scale bar, 50 μm. (d) Immunoblots of SARI and Cp expression in SW480 and HCT116-blank, control and SARI cells. GAPDH was used as a loading control. (e) Western blotting detection of Cp expression of collected nuclear and cytoplasmic proteins. Histone H3 was used as the nucleus loading control and β-actin as the cytoplasm loading control. (f) Immunoblots of Cp and VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. GAPDH was used as the loading control. (g) ELISA detection of VEGF expression in SW480-control and SW480-SARI cells with or without MG132 treatment. (n=4; **P<0.01; Student’s t-test) (h) Measurement of Cp in cell lysates harvested at 0, 2, 4, 6, 8 and 10 h after the addition of cycloheximide (CHX) to arrest protein synthesis. (i) Densitometry analysis of Cp expression in multiple western blots after CHD addition (n=3; **P<0.01; Student’s t-test). (j) Immunoblots of Cp expression in HCT15, HCT15-shNC, HCT15-shSARI-1068 and HCT15-shSARI-554 cells. GAPDH was used as a loading control. (k) Co-immunoprecipitation analysis using anti-HA and anti-CP antibodies of total cell lysates collected from SW480 cells after transfection with HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). Immunoblots of HA and Cp expression. (l) Immunoblots of HA and Cp expression in SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids). GAPDH was used as a loading control. (m) ELISA detection of VEGF expression in the conditional medium from the SW480 cells after transfection with pVax, HA-SARI (encoding 1–79 amino acids), HA-SARI (encoding 80–274 amino acids) and HA-SARI (encoding 1–274 amino acids) with or without TM (4.0 μM) treatment. (n=3; **P<0.01; analysis of variance analysis). IB, immuoblot.

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