Co-immunoprecipitation and subcellular localization of Rab9 and B5 protein with p37-GFP expressed from vvF13LGFP or an ST-246 resistant VV virus variant. (A) BSC-40 cells were infected with wild type VV (left) or an ST-246-resistant variant that exhibits reduced susceptibility to ST-246 (right) in the presence or absence of 10 μM compound. The membrane fractions were extracted as described in Materials and Methods, resuspended in hypotonic buffer and immunoprecipitated with anti-GFP polyclonal antibody. Blots were probed with Rab9 antibody or antiserum to B5 as indicated. Top row: input controls; (B) Quantitative comparison of immunoblot intensity in the absence and presence of ST-246; (C) Cell monolayers were grown on chamber slides and infected with 8 PFU/cell of an ST-246-resistant variant in the presence or absence of 10 μM ST-246. At 14–16 hpi cells were fixed in 4% paraformaldehyde, permeablized with 0.2% TritonX-100 and stained for 1 hour with anti-p230 antibody or anti-CI-MPR antibody. Proteins were visualized using Alexa 594-conjugated secondary antibody. Samples were mounted in ProLong Gold Antifade Reagent (Invitrogen Molecular Probes) containing DAPI for nuclear staining and analyzed using a Zeiss LSM 510 confocal laser-scanning microscope. Images are representative of experiments in which a minimum of 50 infected cells displaying a similar fluorescent phenotype were observed. Images were collected and processed using LSM 510 acquisition and Adobe Photoshop software. Bar, 5 μm.