Identification of Nef region interacting with ASK1.
(A) Using Nef core domain (57-207 aa) sequences, two distinct Nef truncations were designed for mammalian two hybrid studies by successively deleting 33 aa form C-terminal and subsequent cloning in pACT vector so as to obtain Nef (57-174 aa), Nef (57-141 aa) fragments. The Nef core domain (57-207 aa) served as positive control. (B) The HEK-293 cells when co-transfected with ASK1 (1-1051aa) fragment and either of Nef fragments viz. Nef (57-207 aa), Nef (57-174 aa), Nef (57-141 aa) and pG5LUC vector showed 152.56 ,81.68 ,and 1.9 fold increase in expression of luciferase/renilla over pACT-pBIND negative control vector respectively. The data are means +SEM from three distinct experiments. P values of <0.05 were obtained (two-tailed paired t test) when luciferase/renilla expression from ASK1 fragments and Nef co-transfected cell were compared with negative control pACT-pBIND.