Fig. 6. (A) Schematic diagram of -actinin-EGFP fusion monomers. The WT, N and C -actinins are expressed as C-terminal chimeras with EGFP. The primary sequence of the WT -actinin consists of the N-terminal conservative actin-binding region (residues 1-245), a central domain of four spectrin-like repeats implicated in dimerization of -actinin and interaction with other proteins including -catenin (residues 246-712), and the C-terminal domain containing the vinculin-binding site (residues 713-749) and the two EF-hand binding Ca²⁺ motifs (residues 770-817). Numbering of residues includes the initiating methionine. (B) Expression levels of -actinin-EGFP fusion proteins in MCF-7/IGF-IR/WT cells. Expression of EGFP was detected in transiently transfected MCF-7/IGF-IR/WT cells by western blotting of 20 µg of total protein with an antibody against GFP. Upper panels, the representative western blot shows the levels of exogenous EGFP (27 kDa) and -actinin-EGFP chimeras (WT, 130 kDa, N, 104 kDa, C, 109 kDa) expressed as transgenes 48 hours after transfection (asterisks indicate the position of chimera). Lower panel, the same blot was stripped and re-probed with an antibody against -actinin to detect endogenous (100 kDa) and exogenous EGFP--actinins whose positions on the blot are indicated by asterisks. Note that the endogenous -actinin and N -actinin-EGFP overrun in a 100 kDa band. (C) Intracellular localization of WT, N, C -actinin-EGFP fusion proteins in serum-starved and IGF-I-stimulated MCF-7/IGF-IR/WT cells. MCF-7/IGF-IR WT cells were transfected with plasmid encoding WT or N or C -actinin-EGFP. 24 hours after transfection, serum-starved cells were either fixed in 3.7% formaldehyde (a-c) or stimulated with 50 ng/ml IGF-I for 15 minutes and then fixed (d-f). The representative images of the middle optical sections acquired by confocal laser-scanning microscopy show (top panel, a-c) the WT and C -actinins incorporated into the mature cell-cell junctions (pointed by arrow) and N -actinin stained in the cytoplasm and nucleus. Lower panel, d-f: the images show the formation of -actinin-EGFP-containing spikes (arrows in d); diffuse staining for N -actinin-EGFP (e), aggregation of C -actinin-EGFP and formation of the defective microspikes (f). Bar, 10 µm.