FIG. 4.
Processing of GerD-FLAG in spores that lack Lgt. Spores of strains PP23 (gerD lgt amyE::PsspB-gerD-flag), PP17 (gerD amyE::gerD-flag), and PP19 (gerD amyE::PsspB-gerD-flag) were disrupted, and total extracts were prepared as described in Materials and Methods. Spores of strain PP19 were also germinated for 45 min, harvested, purified, lyophilized, disrupted, and extracted as described in Materials and Methods. Aliquots of extracts from equal amounts of spores (except for PP17 samples, where twice as much was run) were run by SDS-PAGE, proteins were transferred to a PVDF membrane, and GerD-FLAG was detected with anti-FLAG antibody. Samples of total extract run in the various lanes were from spores of strains PP23 (dormant) (1), PP17 (dormant) (2), PP19 (germinated) (3), and PP19 (dormant) (4). The numbered lines to the left of the figure denote the migration positions of molecular mass markers, in kDa, and the arrowheads to the right indicate the predicted migration positions of both processed (P) and unprocessed (U) GerD-FLAG monomers (M), dimers (D), and trimers (T). Note that lanes 1 and 2 and lanes 3 and 4 are from different gels and that although extract from twice the amount of spores from strain PP17 was run in this experiment, the bands from the strains appear to exhibit approximately the same intensity. However, in this experiment, the amount of extract analyzed was very high, probably saturating the assay. Also note the gel artifact in lane 3 at the monomer weight (the band curves up slightly).