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. 2009 Jun;174(6):2061–2072. doi: 10.2353/ajpath.2009.080960

Figure 4.

Figure 4

Effect of ADAM10, CXCL16 and ADAM17 knockdown on the constitutive and IFN-γ-induced CXCL16 shedding. A: Western blot analysis of CXCL16 and ADAM10 expression after the transfection of specific siRNAs in the presence or absence of IFN-γ. β-Actin was used as a loading control. B: The amount of cellular CXCL16 was measured after specific siRNA transfection in the presence or absence of IFN-γ by a CXCL16-specific ELISA. All data were reproduced in three independent experiments and are represented as mean ± SE (n = 3). *P < 0.05 considered statistically significant compared with mock or sc-siRNA- transfected cells. #P < 0.05 considered statistically significant compared with sc siRNA transfected and IFN-γ treated cells. C: Human podocytes were transfected with CXCL16 and ADAM10-specific siRNA in the presence or absence of IFN-γ and supernatants were harvested. Supernatants were concentrated by centrifugation and soluble CXCL16 was measured by a CXCL16-specific ELISA. All data were reproduced in three independent experiments and are represented as mean ± SE (n = 3). *P < 0.05 considered statistically significant compared with the mock transfected cells. #P < 0.05, ##P < 0.01 considered statistically significant compared with sc-siRNA-transfected cells. §P < 0.05, §§P < 0.01 considered statistically significant compared with sc siRNA-transfected cells in the presence of IFN-γ. D: Influence of ADAM10 knockdown on the release of CXCL16 in the presence of TNF-α with or without preincubation with 3 μmol/L GW280264X (GW) or 50 μmol/L GM6001(GM). All data were reproduced in three independent experiments and are represented as mean ± SE (n = 3). *P < 0.05 considered statistically significant compared with the sc-siRNA-transfected cells. #P < 0.05 considered statistically significant compared with A10 siRNA-transfected cells. §§P < 0.01 considered statistically significant compared with A10 siRNA-transfected cells in the presence of TNF-α. E: Soluble CXCL16 was measured with a CXCL16-specific ELISA after single or double knockdown of ADAM10 and ADAM17 in the presence or absence of IFN-γ. All data were reproduced in three independent experiments and are represented as mean ± SE (n = 3). *P < 0.05 considered statistically significant compared with the sc-siRNA-transfected cells. ##P < 0.01 considered statistically significant compared with the A10 siRNA-transfected podocytes. §P < 0.05 considered statistically significant to A10 siRNA-transfected cells in the presence of IFN-γ. F: Western blot analysis with ADAM10- and ADAM17-specific antibodies in lysates of podocytes transfected with specific siRNAs in the presence and absence of IFN-γ. ß-Actin Western blot has been performed to demonstrate equal loading. A10, ADAM10; C16, CXCL16; A17, ADAM17; sc, scrambled.