(A) TNF-induced PCD of ABIN-1+/+ and ABIN-1−/− MEF (p < 0.01 between ABIN-1+/+ and ABIN-1−/− cells; means and standard deviations indicated, n=3). (B) TNF-induced caspase-3 and BID expression in ABIN-1+/+ (left) and ABIN-1−/− (right) MEFs. Cleaved, active forms of Bid (tBid) and caspase 3 indicated by arrows. Actin expression shown below as protein loading control. (C) TNF-induced PCD of ABIN-1−/− 3T3s expressing GFP and FLAG-ABIN-1 (black columns on right) or GFP alone (white columns on left), after treatment with the indicated agents. Means and standard deviations indicated, n=3. (D) TNF-induced caspase-8, caspase 3, and BID expression in ABIN-1−/− 3T3s expressing GFP and FLAG-ABIN-1 (left) or GFP alone (right). Cleaved, active forms of caspase 8, caspase 3 and Bid (tBid) indicated by arrows. Actin expression shown below as control. (E) TNF-induced expression of MAP kinase signaling proteins and A20 in ABIN-1−/− and control MEFs. (F) TNF-induced recruitment of RIP1 and FADD to Myc (ABIN-1). Expression of Myc-ABIN-1 in IPs, and expression of FADD and caspase 8 in whole cell lysates (“input”) shown below as controls. (G) TNF-induced recruitment of RIP1, FADD and A20 to caspase 8 in ABIN-1 deficient and control HT1080 cells. Expression of caspase 8 in IPs and expression of ABIN-1, FADD and actin in whole cell lysates (“input”) shown below as controls. All data are representative of 3–5 independent experiments.