Figure 7
TNF and UV light increase the binding of Zfra with WOX1, JNK1 and NF-κB, but weakly with p53 in SK-N-SH cells. (A) Exposure of SK-N-SH cells to TNF (50 ng/ml) for 40 min resulted in upregulation of Zfra and degradation of IκBα. (B) In GST pull-down analysis using SK-N-SH cells, TNF increased the binding of GST-Zfra with NF-κB (p65) and JNK1 (greater than 50%), and weakly with p53 but not IκBα. GST-Zfra physically interacted with endogenous Zfra and WOX1, and TNF limitedly increased the binding (less than 30%). In negative controls, GST alone could not bind the above-indicated proteins. The relative amounts of GST and GST-Zfra used in the pull down are shown. One-twentieth amounts of protein input for endogenous Zfra and other indicated proteins are shown in Western blotting. (C) Similarly, during a 10-min treatment, TNF increased the binding of GST-Zfra with p-WOX1, but not with FADD, RIP, IκBα and Fas. (D) SK-N-SH cells were exposed to UV light (120 mJoule/cm2) and then cultured for 1 hr, followed by processing GST pull-down analysis. UV light increased the binding of endogenous Zfra with p-WOX1 and JNK1. (E) By co-immunoprecipitation, UV light (120 mJoule/cm2) increased the binding of endogenous Zfra with itself and p-JNK1 (at Thr183/Tyr185) (greater than 50% increase), but limitedly with WOX1 (less than 20% increase), in SK-N-SH cells.