Figure 9

Membrane association of BVDV NS4B protein. A. BHK-21 cells were transfected with NS4B-GFP or GFP construct. At 48 h p.t., three hundred micrograms of cell extract were subjected to membrane floatation, followed by western blot with GFP-specific antibody. Lysate refers to crude lysate. Lanes 1 to 4: membrane fractions and lanes 5 to 8: soluble fractions. B. and C. Effect of detergent, high salt or high pH treatment on membrane localization of BVDV NS4B protein. BHK-21 cells were transfected with NS4B-GFP as described above. Three hundred micrograms of cell extract were mixed with (B) 1 m sodium chloride and (C) 0.5% TX-100 or 0.1 M sodium carbonate, pH 11.5. After incubation at 4°C for 30 min, the samples were subjected to membrane floatation followed by immunobloting with GFP-, calnexin- or GAPDH-specific antibody. Notice that only TX-100 treatment redistributes NS4B-GFP protein into the soluble fraction represented by lanes 4 through 8. D. Subcellular distribution of NS4B protein. BHK-21 cells were transfected with NS4B-GFP or GFP construct. At 48 h p.t., the cell extracts were separated into nuclear, mitochondrial microsomal and cytosolic fractions followed by immunobloting with GFP- specific antibody. Notice that NS4B-GFP is more prominent in nuclear and mitochondrial fractions whereas GFP is mostly found in cytosolic fractions.