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Fig. 7. Hic-5 associates and localizes with CRP2 to actin stress fibers. (A) Flag-tagged CRP2 was expressed with HA-tagged Hic-5 wild-type and mutants in COS7 cells: lane 1, wild-type; lane 2, N-paxillin/C-Hic; lane 3, N-Hic/C-paxillin; lane 4, paxillin. 24 hours after transfection, the cells were lysed and subjected to immunoprecipitation with polyclonal anti-HA antibody (lane H) or normal rabbit IgG (lane C) as a control, followed by immunoblotting with monoclonal anti-HA (top) or anti-Flag (M2) (bottom) antibodies. (B) The wild-type and chimeric proteins of Hic-5 and paxillin studied in A. (C) Hic-5 (pCG-LD1mhic-5) and CRP2 (Flag-CRP2) co-expressed in mouse embryo fibroblast cells were co-immunostained using the monoclonal anti-HA (12CA5) and polyclonal anti-Flag antibodies after being exposed to the cyclic stretching. The top, middle and bottom images show Hic-5, CRP2 and a merged image, respectively. The arrow indicates the direction of stretching. Bar, 2 µm. (D) Immunogold electron microscopy of a mouse aorta. (top) L, lumen; ET, endothelial cell; EL, elastic lamina; SMC, smooth-muscle cell. (bottom) Magnified image of a part of the smooth-muscle cell (framed rectangle at top). Arrows indicate immunogold (10 nm) labeling of Hic-5 and arrowheads indicate immunogold (15 nm) labeling of CRP2. (middle) A similar field exposed to control serum. Bars, 2 µm (top) and 100 nm (middle and bottom).





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