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. 2018 Jun 26;115(28):E6604–E6613. doi: 10.1073/pnas.1720125115

Fig. 3.

Fig. 3.

DENV2 induces expression of Tsp29Fb, a putative EV-enriched marker on mosquito cells. (A) qRT-PCR analysis showing expression of A. aegypti Tsp29Fb in uninfected (UI) or DENV2-infected (I) whole mosquitoes at 24, 48, and 72 hpi (n = 4), or in EVs (B) isolated from an uninfected (UI) or DENV2-infected (MOI 5) in vitro cell line or in Aag-2 whole-cell lysates at 48 and 72 hpi (n = 4, in both EVs or cell-lysate panels) (C). qRT-PCR analysis showing expression of A. albopictus Tsp29Fb ortholog in EVs (D) isolated from uninfected (UI) or DENV2-infected (MOI 5) C6/36 cells or in whole-cell lysates (E) at 24, 48, and 72 hpi (n = 8, in both EVs or cell-lysate panels). Tsp29Fb or DENV2 capsid mRNA levels were normalized to mosquito actin levels. P value determined by Student’s two-tail t test is shown. (F) Immunoblotting analysis showing levels of Tsp29Fb and viral E-protein in EVs derived from uninfected (UI) or DENV2-infected (MOI 5) C6/36 cells or in whole-cell lysate at 24-, 48-, and 72-hpi time points. (G) Immunoblotting analysis showing levels of Tsp29Fb and viral E-protein in EVs derived from uninfected (UI) or DENV2-infected (MOI 5, 72 hpi) C6/36 cells or in whole-cell lysates upon DENV2 infection with different viral doses (MOI 1–5) is shown. Immunoblotting analysis of CD63 (Tsp29Fb) IP proteins in RIPA (H) or in 1× PBS buffer (I) with 4G2 antibody showing detection of viral E-protein in EVs or whole-cells lysates derived from DENV2-infected (MOI 5, 72 hpi) C6/36 cells. Uninfected cells were used as controls in all assays. Gel images showing total protein profiles in F–I serve as loading control for respective immunoblots. Tsp29Fb and DENV2 E-protein molecular mass is indicated as kilodaltons.