Skip to main content
. 2003 Feb;77(3):1691–1702. doi: 10.1128/JVI.77.3.1691-1702.2003

FIG. 3.

FIG. 3.

Membrane association, palmitoylation, and phosphorylation of polyproteins. (A) Bac12CA3-infected Tn5 cells were labeled as for Fig. 2E. Postnuclear supernatant was subjected to flotation in a discontinuous sucrose (weight/weight) gradient consisting of 0.675 ml of 10%, 3.5 ml of 50%, and 0.625 ml of 60% sucrose on top of a 0.5-ml 67% sucrose cushion. Fractions of 0.5 ml were collected from the top and analyzed by immunoblotting with antiserum against nsP1. The sample was initially included in the 60% sucrose layer (fractions 8 and 9), and upon centrifugation, membrane-bound proteins floated to the 10 to 50% sucrose interface (fractions 2 and 3). (B) Fractions 2 and 3 were pooled, and aliquots were subjected to treatments with 50 mM Tris-HCl (pH 7.5)-100 mM NaCl (TN buffer), 1 M NaCl in TN buffer, and 50 mM Na2CO3 at pH 12. After 30 min of incubation on ice, the samples were centrifuged at 70,000 × g for 30 min. The pellet (P) and supernatant (S) fractions were immunoprecipitated with anti-nsP3 antiserum, followed by SDS-PAGE in a 7.5% gel and autoradiography. (C) Palmitoylation of SFV P12CA3. Tn5 cells were infected with Bac12CA3 and labeled at 44 h p.i. with 300 to 400 μCi of [9,10(n)-3H]palmitic acid for 8 h. Bac123- and Bac1-infected and mock-infected cells served as controls. Cells were collected, the P15 fraction was prepared, and protein expression was verified by immunoblotting (WB) (lanes 1 and 2) with anti-nsP1 antibody. Part of the P15 fraction was immunoprecipitated by anti-nsP1 antiserum. Samples were analyzed with SDS-PAGE in a 7.5% gel, and incorporated [3H]palmitate was visualized with a PhosphorImager. (D) In vivo phosphorylation of SFV P12CA34, P12CA3, and P2CA3 proteins. Tn5 cells were infected with Bac12CA34, Bac12CA3, or Bac2CA3, labeled with [32P]orthophosphate at 41 to 44 h p.i., harvested, and lysed by boiling in 1% SDS. Proteins were immunoprecipitated with anti-nsP3 antibody and analyzed with SDS-PAGE and a PhosphorImager. In all panels, positions of proteins are indicated on the right and those of molecular weight (Mw) markers, in thousands, are indicated on the left.