FIG. 3.

Sucrose gradient fractionation of a crude extract of GFLV-infected T-BY2 protoplasts harvested at 72 hpi. Clarified crude extracts prepared from 10⁶ protoplasts were fractionated by a 4-h centrifugation at 29,000 rpm through an 11-ml linear 20 to 45% sucrose gradient containing 5 mM MgCl₂. Sixteen fractions (670 μl each) were collected. (A) For Western blot analysis, 15-μl aliquots of each fraction were analyzed by SDS-12.5% PAGE, electrotransferred onto an Immobilon-P membrane, and then further immunoreacted with anti-VPg antibodies to reveal VPg-containing proteins. The molecular mass of protein standards are indicated in kilodaltons on the right side, and the apparent molecular masses of the VPg precursors are shown on the left side. (B and C) For dot blot assays, 5-μl aliquots of each fraction were applied as dots on two strips of Immobilon-P membrane (Millipore). The strips were treated either with JIM84 (B) or with anti-HDEL monoclonal antibodies (C). The membranes were then incubated with goat anti-rabbit IgG (A) or goat anti-mouse IgG (B and C) coupled to horseradish peroxidase and revealed by chemiluminescence.