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. 2008 Apr 2;82(13):6272–6287. doi: 10.1128/JVI.02345-07

FIG. 6.

FIG. 6.

FIG. 6.

FIG. 6.

Iodixanol density gradient fractionation of HCMV-infected HFF cells transfected with pp28 mutants. HFF cells were electroporated with approximately 5 μg of expression plasmids encoding pp28Mut61-EGFP or pp28Mut35-EGFP and infected 2 days later with HCMV at an MOI of 0.1. The cells were harvested at day 6 postinfection. (A) Expression of multimeric forms of pp28-EGFP fusion proteins and viral pp28. PNSs were prepared from the harvested cells, solubilized under lower concentrations of SDS (0.1% SDS) and nonreducing conditions, subjected to SDS-PAGE in 10% gel, and detected with anti-pp28 MAb and anti-EGFP polyclonal antibodies by immunoblotting. Note the faster migration of dimeric forms of pp28Mut35-EGFP compared to dimeric form of viral pp28, even though the predicted mass (in kilodaltons) from the amino acid sequence of pp28Mut35-EGFP is greater than that of viral pp28. PNS from cells infected with HCMV was prepared as a control. (B and C) Analysis of gradient fractions by immunoblotting. PNS were sedimented by centrifugation through iodixanol gradients as described in Materials and Methods. The gradient was fractionated by removing 1-ml fractions from the top. Thus, fraction 1 represents the top of the gradient, and fraction 10 represents the bottom of the gradient. Gradient fractions were analyzed by Western blotting. Proteins were detected with an antibody specific for pp28 or EGFP (top) as described in Materials and Methods and analyzed by densitometry (middle and bottom). The average density of the signal from protein in each gradient fraction was measured and plotted to provide graphic comparison of the pattern of fractionation of virus-encoded pp28 to those of pp28Mut61-EGFP (B) and pp28Mut35-EGFP (C) in monomeric forms (top) and in dimeric forms (bottom). Vpp28, GFP61, and GFP35 indicate viral pp28, pp28Mut61-EGFP, and pp28Mut35-EGFP, respectively. 1x and 2x indicate monomer and dimer, respectively. An asterisk (*) and two asterisks (**) indicate viral pp28 fractionated from HCMV-infected HFF cells that had been transfected with pp28Mut61-EGFP and pp28Mut35-EGFP, respectively. The membranes were stripped and reprobed with anti-CD63 MAb to identify gradient fractions containing endosomes (40).