Figure 4.

Subcellular Localization of BeGC1 Protein

(A) Western blot analysis of subcellular fractions of zoospore lysates obtained by differential centrifugation, as described in the Supplemental Experimental Procedures. Fractions were resolved through SDS-PAGE followed by western blotting and were developed using rabbit antisera against BeGC1, BePAT1, and α-tubulin, as well as the fluorescent CF680 Goat anti-rabbit IgG as a secondary antibody. The bound complexes were detected using the Odyssey Infrared Imaging System.

(B) Localization of BeGC1 by immunofluorescence microscopy. Zoospores were fixed with 4% p-formaldehyde and 1% calcium chloride, permeabilized with PBS containing 0.1% Triton X-100, and incubated with rabbit anti-BeGC1 antiserum. The reactivity was developed with a specific goat anti-rabbit IgG antibody conjugated with Alexa-Fluor 488 (Molecular Probes). The lipid droplets of the eyespot were visualized with the lipid-specific fluorescent dye Nile Red. From top left to bottom right, the following are shown: zoospore under phase contrast (differential interference contrast image), BeGC1 (green), lipid droplets (red) of the eyespot, and a merge of BeGC1 and lipid droplets images. The arrows indicate the position of the eyespot apparatus, and the arrowhead shows the zoospore flagellum. The images shown are at 1000× magnification.