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Fig. 2. The actin-binding property of SWAP-70. (A) Binding of full-length SWAP-70 to F-actin. His–SWAP-70 or His–SWAP-70(1-564), lacking the actin-binding domain, was mixed with (+) or without (–) 10 µM polymerized non-muscle actin. Proteins in the supernatant (S) and the pellet (P) were analyzed by SDS-PAGE followed by Coomassie Blue staining. The band seen at the position of actin in the lane of actin (–) is the degradation product of His–SWAP-70. The same protein may be present in the lane of actin (+). (B) Quantitative analysis for binding of His–SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing of 5 µM polymerized non-muscle actin with various amounts of His–SWAP-70 (0 µM to 5 µM). (C) Binding property of full-length SWAP-70. A co-sedimentation assay was performed by mixing of His–SWAP-70 with polymerized 10 µM actin derived from human platelets (non-muscle) or rabbit skeletal muscle (muscle). (D) Binding of the PH domain of SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing of His–SWAP-70 PH with polymerized 10 µM muscle actin, non-muscle actin, or without (–) actin. (E) Quantitative analysis for binding of the PH domain of SWAP-70 to F-actin. A co-sedimentation assay was performed by mixing 5 µM polymerized non-muscle actin with various amounts of His–SWAP-70 PH (0 µM to 5 µM). Quantitative analysis was done as in B. (F) Actin-binding properties of the various mutants of SWAP-70. The structure of SWAP-70 and its truncation mutants is shown. All constructs were tagged with His at the N-terminus. The actin-binding property of each protein is summarized on the right.
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