Figure 1. The p75–ErbB1 KD associates with ErbB2 and inhibits NRG-dependent activation of ErbB2 in 293 cells. A, Experimental strategy of the p75–ErbB1 KD construct. The p75–ErbB1 KD would heterodimerize with ErbB receptors and inhibit tyrosine phosphorylation of the associated ErbB receptors. The p75–ErbB1 KD itself does not bind EGF, because the ligand-binding domain is replaced with the corresponding domain of p75. B, The p75–ErbB1 KD does not affect TrkA signaling in the presence of NGF. 293T cells were transfected with TrkA, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment with NGF, the lysates were immunoprecipitated with pan-Trk antibody, and the activation status of TrkA was detected using 4G10 antibody. The same blot was reprobed for the presence of the transfected TrkA using pan-Trk antibody in IP/W. Note that tyrosine phosphorylation of TrkA is not affected in the presence of the p75–ErbB1 KD. The presence of the p75–ErbB1 KD is shown as an HA blot. C, The p75–ErbB1 KD associates only with ErbB2, but not with ErbB3 or ErbB4 in 293T cells. 293T cells were transfected with ErbB2, 3, or 4, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After immunoprecipitation with antibodies against ErbB2, 3, or 4, or HA tag included in the p75–ErbB1 KD, the associated p75–ErbB1 KD was detected with anti-HA antibody. A portion of the lysates was subjected in parallel to IP/W, using ErbB2, 3, or 4 antibodies as controls. D, The p75–ErbB1 KD inhibits NRG-dependent activation of ErbB2. 293T cells were transfected with ErbB4 to facilitate activation of the endogenous ErbB2 with NRG, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment of soluble NRG, the lysates were immunoprecipitated with ErbB2 antibody and the activation status of ErbB2 was detected using PY99 antibody. The presence of the p75–ErbB1 KD was detected with HA antibody in IP/W. Note that tyrosine phosphorylation of ErbB4 is also reduced in the presence of the p75–ErbB1 KD. E, ErbB4 is present in the p75–ErbB1 KD–ErbB2 complex. 293T cells were transfected with Flag–ErbB4 to facilitate activation of the endogenous ErbB2 with NRG, and subsequently infected with the control GFP or p75–ErbB1 KD adenovirus. After 5 min of treatment of soluble NRG, the lysates were immunoprecipitated with ErbB2 antibody, and the presence of ErbB4 and the p75–ErbB1 KD was probed with anti-Flag or HA antibodies. W:PY, Phosphotyrosine.