Attachment of ubiquitin to proteins represents a central mechanism for the 
regulation of protein metabolism and function. In the NF-kappaB pathway, binding 
of NEMO to polyubiquitinated substrates initiates the pathway in response to 
cellular stimuli. Other polyubiquitin binding proteins can antagonize this 
pathway by competing with NEMO for polyubiquitin. We have used protein arrays to 
identify polyubiquitin binding proteins that regulate NF-kappaB activity. Using 
polyubiquitin as bait, protein arrays were screened and polyubiquitin binders 
identified. Novel polyubiquitin binders AWP1, CALCOCO2, N4BP1, RIO3, TEX27, 
TTC3, UBFD1 and ZNF313 were identified using this approach, while known 
NF-kappaB regulators including NEMO, A20, ABIN-1, ABIN-2, optineurin and p62 
were also identified. Overexpressed AWP1 and RIO3 repressed NF-kappaB activity 
in a manner similar to optineurin, while siRNAs directed against AWP1 and RIO3 
also reduced NF-kappaB activity. TNFalpha-dependent degradation of IkappaBalpha 
was also suppressed by overexpression of AWP1 and RIO3, possibly due to the 
polyubiquitin binding activity of these proteins. Protein array screening using 
polyubiquitin enabled rapid identification of many known and novel polyubiquitin 
binding proteins and the identification of novel NF-kappaB regulators.