Processing of baculovirus-encoded DSP-PP₂₄₀ in conditioned medium requires an activity that is only secreted by Sf9 cells early but not late after infection.

<p>(A) Processing of DSP-PP₂₄₀ in conditioned medium of virus-infected Sf9 cells. Sf9 cells were infected with baculovirus containing the DSP-PP₂₄₀ cDNA. In lane 1, 3 days after infection, CM_0–3d was collected and processed for native PAGE and Stains-All staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. In lane 2, CM_0–3d was incubated for an additional 3 days at 28°C before processing for PAGE. M represents protein size markers. (B) Medium conditioned by infected cells 3 to 7 days after infection lacks processing activity. 3 days after infection of Sf9 cells with baculovirus containing DSP-PP_240, the medium was replaced with fresh Grace’s medium containing 10% FBS and 50 µg/ml Gentamycin and the cells were cultured for an additional 4 days. In lane 1, the resulting conditioned medium, CM_3–7d was collected and processed for native PAGE and Stains-All staining. In lane 2, CM_3–7d was incubated for an additional 3 days at 28°C before processing. M represents protein size markers. (C) Quantification of DSP-PP₂₄₀ precursor processing. Using NIH J image, we measured the image intensity of DSP-PP₂₄₀ and PP₂₄₀ bands in gels shown in panels A & B and two other similar gels. Calculation of percent processing was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. Because DSP staining was very weak by Stains-All staining, total density was defined as the sum of the DSP-PP₂₄₀ and PP₂₄₀ image densities. Numbers 1–4, respectively, refer to panel A lane 1, panel A lane 2, panel B lane 1 and panel B lane 2. Error bars represent standard deviation of at least duplicate samples.