FIG. 2.

(A) Flow cytometry analysis of baculovirus-infected Sf9 cells producing mGFP in the absence (red plot) or presence (green plot) of bacterial chaperones DnaK and DnaJ. Sf9 cell cultures were set up at a density of 2 × 10⁶ cells/ml, supplemented with 5% fetal calf serum, and infected at an MOI of 2. Cells were harvested at 72 hpi and rinsed with cold phosphate-buffered saline (PBS). Flow cytometry analyses were performed with intact cells resuspended in PBS on a FACSCalibur system (Becton Dickinson). The fluorescence emission in the FL1 channel was analyzed using WinMDI 2.9 software. Uninfected cells (black plot) were used as a negative control, and measurements were recorded for three independent replicas. (B) Protein amounts in total, soluble, and insoluble cell fractions in the absence (red bars) or presence (green bars) of bacterial chaperones DnaK and DnaJ were estimated by Western blot analysis after disruption of cells harvested at 72 hpi. Sf9 cells were disrupted in lysis buffer (extraction buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA] containing 1% Tergitol NP-9 [Sigma] and Roche's protease inhibitor cocktail [catalog no. 11836170001]) on ice for 30 min and Dounce homogenized. Cell lysates were treated with DNase (25 μg/ml) and MgSO₄ (10 mM), and soluble and insoluble cell fractions were separated by centrifugation at 9,500 × g for 10 min. The insoluble cell pellet was washed with extraction buffer containing 0.5% Triton X-100 and resuspended in extraction buffer. The inset box shows the estimated half-life for mGFP in the absence (red bars) or presence (green bars) of DnaKJ after protein synthesis arrest at 24 hpi by the addition of cycloheximide at a final concentration of 100 μg/ml. Western blot analysis of a time course experiment showing mGFP production in Sf9 cells at different hpi is depicted on the inset graph. Material from the same number of cells was loaded into the gels for protein determination. (C) Specific fluorescence of mGFP produced in the absence (red bars) or presence (green bars) of bacterial chaperones DnaK and DnaJ at 72 hpi. Fluorescence emissions of lysates and soluble and insoluble cell fractions were measured in triplicate, with no further treatment, using a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.). Fluorescence data were combined with protein amounts to obtain the specific fluorescence of mGFP, defined as fluorescence units per μg of mGFP. The significance of the differences between data values determined in the absence and in the presence of DnaK/DnaJ is indicated through P values from an analysis of variance test in panels B and C.