AIP1 mediates TNF-α–induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3
J. Clin. Invest. Rong Zhang, et al. 111:1933 doi:10.1172/JCI17790 [Go to this article.]

Figure 3
TNF-α induces association of AIP1 with ASK1 in ECs. (a) BAECs were transfected with AIP1-F or AIP1-N followed by treatment with TNF-α (10 ng/ml for 15 minutes). Cell lysates were immunoprecipitated with anti-ASK1, and ASK1 in the immunoprecipitate was determined by Western blot with anti-AIP1. (b) Specificity of AIP1 antibody. A polyclonal antibody against AIP1 was produced by Cocalico Biologicals Inc. by immunizing rabbits with GST–AIP1-PH. 293T cell lysates expressing FLAG-tagged AIP1-F, -N, and -C were used to determine the specificity of anti-AIP1 by Western blot (lanes 1–3). Anti-FLAG was used as a control (lanes 4–6). (c) AIP1 is highly expressed in cultured ECs. AIP1 expression in cell lysates from HUVECs, breast cancer MCF-7 cells, or prostate cancer cell line LNCaP (20 μg of total protein from each sample) was measured by Western blot with anti-AIP1. (d) TNF-α induces association of AIP1 with ASK1, whereas it dissociates 14-3-3 from ASK1. HUVECs were either untreated or treated with TNF-α (10 ng/ml for 15 minutes). Cell lysates were immunoprecipitated with anti-ASK1 followed by Western blot with anti-AIP1 or anti-14-3-3. (e) TRAF2 induces association of AIP1 with ASK1. BAECs were transfected with vector control (VC) or FLAG-tagged TRAF2 (TR2). Association of TRAF2 or AIP1 with ASK1 was determined by immunoprecipitation with anti-ASK1, followed by Western blot with anti-FLAG or anti-AIP1. (f) AIP1 has no effect on TRAF2-ASK1 complex. BAECs were transfected with vector control or AIP1-N, followed by TNF-α treatment (10 ng/ml). Endogenous TRAF2-ASK1 complex was determined by immunoprecipitation with anti-TRAF2 followed by Western blot with anti-ASK1. IP, immunoprecipitate; IB immunoblot.