TI - TNF induces the binding of Zfra with Tyr33 -PHOSphorylated WOX1 , JNK1 and NF-kappaB . AB - To verify the above observations , we utilized GST ( Glutathione S-Transferase ) pull-down analysis and co-immunoprecipitation to characterize the binding of Zfra with its partners . Zfra was tagged with GST at its N-terminus . Recombinant GST-Zfra protein was produced in bacteria , and then purified using glutathione-Sepharose 4B resin . Similarly , recombinant GST alone was produced and purified . Exposure of Zfra-sensitive SK-N-SH cells to TNF resulted in an increased expression of Zfra and degradation of IkappaBalpha (Figure 7A) , indicating that this cell line has a functional TNF signal pathway . This cell line expresses a low level of Zfra . Zfra is able to undergo self-association and binds JNK1 [10] . In GST pull-down assays , GST-Zfra was shown to interact with endogenous Zfra and WOX1 , and TNF barely increased the binding in SK-N-SH cells (Figure 7B) . In addition , TNF increased the binding of GST-Zfra with JNK1 , NF-kappaB ( p65 ) and p-WOX1 (Tyr33 PHOSphorylated) , and weakly with p53 in a time-related manner ( Figure 7B and 7C ) . Zfra could not interact with IkappaBalpha , FADD , RIP and Fas ( Figure 7B and 7C ) . Similarly , in response to UV light ( 120 mJoule/cm2 ) , there was an increased binding of GST-Zfra with endogenous Zfra , p-WOX1 and JNK1 in SK-N-SH cells , as determined by pull-down analysis ( Figure 7D ) . UV light increased the binding of endogenous Zfra with PHOSphorylated JNK1 ( at Thr183/Tyr185 ) in SK-N-SH cells , as determined by co-immunoprecipitation ( Figure 7E ) .