Figure 1.

(A) Association of cytoskeletal proteins of D. discoideum with detergent-extracted cytoskeleton preparations from different developmental stages. Cells were starved in shaking culture (as indicated) and lysed with 1% Triton X-100 in the presence of cytoskeleton-stabilizing buffer (10 mM PIPES, pH 6.8, 20 mM KCl, 2 mM MgSO₄, 30% glycerol). Triton X-100–insoluble material was pelleted and extracted with Ca²⁺-buffer (10 mM PIPES, pH 7.0, 20 mM KCl, 2 mM MgSO₄, 20 mM CaCl₂, protease inhibitors). Proteins of the Ca²⁺ extracts were separated on SDS-PAGE, visualized by Coomassie blue staining, and analyzed for the accumulation of proteins during the transition of vegetative cells (t₀) to highly motile preaggregation stage (t₃ to t₉). The increased association of a minor protein component, a 30 kDa, with the Ca²⁺-extractable fraction of Triton X-100–insoluble material from aggregation-competent cells is shown in panel A by an arrow and is more clearly visible in Western blots with anti-DdLim–specific antibodies (Figure 1B). The exact values indicated in the text were quantified by phospho-image analysis of Western blots using ¹²⁵I-labeled anti-DdLim antibodies.