Co-precipitation of DV with ApoA-I.
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Abstract
<p>(A) Vero cells were infected with DV at a MOI of 1 and culture medium were changed to DMEM with 10% human serum HS (HSM) at 2 dpi. The mock-infected cells by DMEM was used as a control and also subjected to the same medium change. Culture supernatants were harvested at 7 dpi and purified by sucrose cushion ultracentrifugation (UC). The virus pellets were resuspended in serum-free DMEM. Presence of ApoA-I was analyzed by Western blotting using anti-ApoA-I antibody. (B) Human serum was added into DV/SFM to a final concentration of 10% and the mixture was incubated at 4° for 1 hour, followed by sucrose cushion ultracentrifugation. The pellets were analyzed by Western blotting using anti-ApoA-I and anti-E antibodies respectively. (C) Co-immunoprecipitation of ApoA-I with DV. AD-293 cells were transfected with a plasmid expressing FLAG-tagged ApoA-I (pApoAI-FLAG) and cultured in serum-free DMEM. At 3 dpt, secreted ApoA-I in the culture supernatant was purified with anti-FLAG M2 Affinity Gel. The resulting ApoAI-FLAG/M2 beads were washed twice with 1×TBS and incubated with DV/SFM at 4°C for over night. The co-immunoprecipitates were eluted and detected by Western blotting with anti-E and anti-FLAG antibodies. As a control, co-immunoprecipitation was also performed using the supernatant from cells transfected with empty vector p3×FLAG-CMV-14 (pFLAG). M, pre-stained protein marker.</p