Fig 10

(A and B) Examination on the interaction between Env38 and MHC-IIα or MHC-IIβ, in which the Flag-tagged Env38 and HA-tagged MHC-IIα (A) or HA-tagged MHC-IIβ (B) were coexpressed in HEK293T cells, followed by immunoprecipitation with rabbit anti-Flag or rabbit IgG (negative control). Western blot was performed with mouse anti-Flag or anti-HA Ab. (C) Examination on the interaction between leukocyte Env38 and MHC-IIα proteins of zebrafish under SVCV stimulation. The lysates were immunoprecipitated with rabbit anti-MHC-IIα or rabbit IgG. Western blot was performed with mouse anti-Env38 Ab. Similarly, the lysates were immunoprecipitated with mouse anti-Env38 or mouse IgG, and Western blot was performed with rabbit anti-MHC-IIα Ab. (D) Examination on the effect of glycosylation on the interaction between Env38 and MHC-IIα. The Flag-tagged Env38 and HA-tagged MHC-IIα were coexpressed in HEK293T cells under treatment with tunicamycin, followed by immunoprecipitation with rabbit anti-Flag or rabbit IgG. Western blot was performed with mouse anti-Flag or anti-HA Ab. (E) Examination on the interaction between Env38 and CD4, in which the Flag-tagged Env38 and the Myc-tagged CD4 were coexpressed in HEK293T cells, followed by immunoprecipitation with rabbit anti-Flag or rabbit IgG. Western blot was performed with the mouse anti-Flag or anti-Myc Ab. (F) Examination on the interaction between leukocyte Env38 and CD4 proteins of zebrafish under SVCV stimulation. The lysates were immunoprecipitated with rabbit anti-CD4 or rabbit IgG. Western blot was performed with mouse anti-Env38 Ab. Similarly, the lysates were immunoprecipitated with mouse anti-Env38 or mouse IgG, and Western blot was performed with rabbit anti-CD4 Ab. (G) Examination on the interactions between Env38 and CD4 proteins, in which the His-tagged Env38 proteins were purified from Sf9 or E. coli cells and the Myc-tagged CD4 was expressed in HEK293T cells, followed by immunoprecipitation with rabbit anti-Myc or rabbit IgG. Western blot analysis was performed with the mouse anti-Myc or anti-His Ab. (H) Examination on the interactions among Env38, MHC-IIα and CD4, in which the Flag-tagged Env38, HA-tagged MHC-IIα and Myc-tagged CD4 were coexpressed in HEK293T cells, followed by immunoprecipitation with rabbit anti-HA Ab. Western blot was performed with mouse anti-Flag, anti-HA or anti-Myc Ab. (I) Examination on the interactions among EnvTM, EnvSU, CD4 and MHC-IIα, in which the Flag-tagged EnvTM, Flag-tagged EnvSU, Myc-tagged CD4 and HA-tagged MHC-IIα were coexpressed in HEK293T cells in different combinations, followed by immunoprecipitation with rabbit anti-Flag Ab. Western blot was performed with mouse anti-Myc, anti-HA, or anti-Flag Ab. (J) Examination on the interactions among EnvSU, CD4ΔD1, CD4ΔD1-2, CD4ΔD1-3 and MHC-IIαΔα1, in which the Flag-tagged EnvSU, Myc-tagged CD4ΔD1, Myc-tagged CD4ΔD1-2, Myc-tagged CD4ΔD1-3 and HA-tagged MHC-IIαΔα1 were coexpressed in HEK293T cells in different combinations, followed by immunoprecipitation with rabbit anti-Flag Ab. Western blot was performed with mouse anti-Myc, anti-HA, or anti-Flag Ab. The same lysates were simultaneously immunoblotted to monitor the expression of CD4ΔD1, CD4ΔD1-2, CD4ΔD1-3, MHC-IIαΔα1, and EnvSU. In the Co-IP assays, the same lysates were simultaneously immunoblotted to monitor the expression of proteins in input. (K) Functional evaluation on the association among Env38, MHC-IIα and CD4 proteins distributed on MHC-II⁺Env38⁺APCs and CD4⁺ T cells by examining the competitive inhibition of CD4⁺ T cell activation by using a soluble D2 domain protein of CD4 (sCD4-D2, 5 μg/ml and 10 μg/ml). The activation of CD4⁺ T cells was determined by the transcriptional expression levels of cd4, cd154, lck and il-2 genes through RT-qPCR. The RT-qPCRs were run in combination with the endogenous β-actin control. Error bars represented SEM. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference).