Fig. 1. Wild-type and calcium-insensitive forms of the 34 kDa protein. (A) Sequence of wild-type 34 kDa and 34 kDa EF2 proteins. Nucleotide and amino-acid sequences of the wild-type and modified EF hand 34 kDa proteins are shown. The altered nucleotides and amino acids are shown in bold. (B) Expression and purification of the 34 kDa and 34 kDa EF2 proteins. SDS-PAGE gel stained with Coomassie blue. Lanes 1 and 3 were loaded with 100 µg of BL21 (DE3) expressing 34 kDa and 34 kDa EF2 proteins, respectively. Lanes 2 and 4 were loaded with 25 µg of purified 34 kDa and 34 kDa EF2 proteins, respectively. (C) Western blot showing Dictyostelium AX2 and 34-kDa-null cells expressing the wild-type 34 kDa protein and 34 kDa EF2 proteins. Proteins from cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with a monoclonal antibody B2C reactive to the 34 kDa protein.

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