A, GST-MKK7β1 N-terminal truncations tested for JNK1 binding. GST-MKK7-(1–85) contains all three D-sites, 1–60 contains the first two, and 1–38 contains only the first. 35S-Labeled JNK1α1 was produced by in vitro translation; GST pull-down assays were used to test the binding between the truncations and JNK1α1. B, JNK1 (~1 pmol) was tested for binding to 10 and 40 µg of GST (lanes 2 and 3), GST-MKK7-(1–85) (lanes 4 and 5), GST-MKK7-(1–60) (lanes 6 and 7), or GST-MKK7-(1–38) (lanes 8 and 9). Lane 1 shows 10% of the total JNK1 input. The lower panel shows Coomassie Blue (CB) staining of the sedimented GST fusion proteins. C, quantification of JNK1-MKK7 binding, using 40 µg of GST-MKK7 truncations per reaction. Standard error bars are shown (n = 8).