Fig. 2. The D31
A point mutation disrupts the interaction with PINCH and
impairs ILK localization to focal adhesions. (A-C) PINCH binding. Lysates of
C2C12 cells expressing GFP-tagged wild-type or mutant (D31A) form of ILK
were mixed with rabbit anti-GPF antibodies. The GFP-D31A (lane 3) and GFP-ILK
(lane 4) immunoprecipitates were analyzed by western blotting with
HRP-conjugated anti-GFP antibodies (A), rabbit anti-PINCH antibodies and
HRP-conjugated anti-rabbit IgG antibodies (B), or mouse monoclonal
anti-CH-ILKBP antibody 3B5 and HRP-conjugated anti-mouse IgG antibodies (C).
Lanes 1 and 2 were loaded with cell lysates (10 µg/lane) as indicated in
the figure. (D,G) Subcellular localization. C2C12 cells transfected with
expression vectors encoding GFP-D31A (D,E) or GFP-ILK (F,G) were plated on
fibronectin-coated coverslips and stained with a mouse monoclonal
anti-paxillin antibody and a Rhodamine RedTX-conjugated anti-mouse
IgG antibody. GFP-D31A, GFP-ILK and paxillin were visualized under a
fluorescence microscope equipped with GFP (D,F) and rhodamine (E,G) filters.
The experiments were performed twice and similar results were obtained. Bar,
10 µm.