Figure 2

Tat-JBD could inhibit JIP-1–JNK3 interaction in vitro and in vivo. Dissociating effects of Tat peptides on the JIP-1–JNK3 interaction. (a and b) His-JNK3(40–422), His-JNK2(full length) and GST-JIP1(127–282) were expressed and purified. Purified JNK3α1 was preincubated with 10 μM Tat-JBD-4A (final concentration), 10 μM or 20 μM of Tat-JBD (final concentration), whereas JNK2 was preincubated with 10 μM Tat-JBD-4A and 10 μM Tat-JBD (final concentration), respectively. GST–JIP1(127–282) fusion proteins bound to glutathione agarose beads were incubated with above mixtures and eluted with elution buffer. Proteins were analyzed by SDS-PAGE and Coomassie blue R250 staining. Lane 1–3 and 7–8: flow-through, Lane 4–6 and 9–10: elution. 1, JNK3α1+20 μM Tat-JBD 2, JNK3α1+10 μM Tat-JBD 3, JNK3α1+10 μM Tat-JBD-4A 4, JNK3α1+20 μM Tat-JBD 5, JNK3α1+10 μM Tat-JBD 6, JNK3α1+10 μM Tat-JBD-4A. 7, JNK2+10 μM Tat-JBD 8, JNK2+10 μM Tat-JBD-4A 9, JNK2+10 μM Tat-JBD 10, JNK2+10 μM Tat-JBD-4A. (c and d) Co-immunoprecipitation of JIP-1 with JNKs subunits in mouse SNc fractions treated with Tat-JBD. Sample proteins from SNc fractions were immunoprecipitated (IP) with anti-JIP-1 or anti-JNK3 or JNK1/2 antibodies and then blotted (IB) with anti-JNK3 or JNK1/2 antibodies. Quantitative representation of JIP-1 interactions JNK3 or JIP-1 interaction with JNK1/2. Data are expressed as the mean±s.d. and as folds vs ^aP<0.05 vs saline; ^bP<0.05 vs MPTP groups (n=6 mice).