Figure 7.

In vitro high-speed cosedimentation assays revealed that [H⁺] and [Ca²⁺] simultaneously regulated the F-actin affinity of full-length and the N-terminal half of LlLIM1. High-speed (100,000g) F-actin in vitro cosedimentation assay samples were collected from reaction buffers containing 4 μm F-actin and 2 μm LlLIM1 (A–C), 6 μm LlLIM1N (D–F), or 28 μm LlLIM1C (G–I) under different pH conditions (pH 6.25–7, MES buffer; pH 7–8, Tris buffer) without (A, D, and G) or with (B, E, and H) 4 mm EGTA. C, F, and I were samples from reaction buffers containing 4 μm F-actin and 2 μm LlLIM1, 6 μm LlLIM1N, or 28 μm LlLIM1C under pH 6.25 conditions and in the presence of different amounts of EGTA (0–8 mm) used for cosedimentation assays with the estimated [Ca²⁺] indicated. In all experiments, after 1 h of incubation, the samples were centrifuged at 100,000g for 45 min, and the resulting pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE and Coomassie Blue staining. e[Ca²⁺], Free Ca²⁺.