Fig. 3. In vitro binding of MEK1, vinculin and Elk1 to ERK2. 1 µg recombinant GST-ERK2 was incubated with 0.5 µg recombinant His-MEK1, purified vinculin or recombinant Elk-1 (1 h 23°C) in buffer containing 20 mM HEPES PH 7.4, 20mM MgCl2, 20mM NaCl, 1mM DTT and 10 mg/ml of aprotinin), with the indicated concentration of CaCl₂. The beads were washed 3 times with a buffer containing 20 mM HEPES PH 7.4, 20mM MgCl₂, 100mM NaCl and 1mM DTT) with the same CaCl₂ concentrations. Then the beads were boiled in sample buffer, and subjected to Western blotting with anti-MEKs, vinculin and Elk-1 Abs. Total loading was detected by polyclonal anti-GST Ab.