The Pafah1b Complex Interacts with the Reelin Receptor VLDLR (original) (raw)
Figure 1
Pafah1b2 and Pafah1b3 bind VLDLR but not ApoER2. (A) Pafah1b3 binds VLDLR in transfected cells. Pafah1b3-MYC was co-expressed in 293T cells with GFP- (lanes 1–3) or HA-tagged VLDLR (lanes 4–6). VLDLR was co-precipitated with MYC antibodies directed against Pafah1b3 (lanes 2 and 5), but not with control antibodies (lanes 3 and 6). Conversely, Pafah1b3 was co-precipitated with HA (lane 9) or GFP (lane 10) directed against VLDLR, but not with control antibodies (lanes 11–12). Lanes 1 and 4 show VLDLR, and lanes 7–8 show Pafah1b3 in the WCL. Blots were probed with the GFP (lanes 1–3), HA (lanes 4–6) or Myc antibodies (lanes 7–12). (B) Pafah1b2 and Pafah1b3 bind VLDLR in a cell-free-system (TNT). In vitro translated proteins were radiolabeled with 35S and analyzed by SDS-PAGE (lanes 1–4). Individual Pafah1b subunits were combined with equal amounts of VLDLR and immunoprecipitated with FLAG (lanes 5–7) or a negative control antibody (lanes 8). Proteins were detected by autoradiography. Note that VLDLR co-precipitated with Pafah1b α subunits, but not with Lis1. (C) Pafah1b2 and Pafah1b3 do not bind ApoER2 in transfected cells. ApoER2-GFP was co-expressed in 293T cells with the indicated proteins, and co-immunoprecipitated with HA antibodies directed against Dab1 (lane 1). FLAG antibodies directed against the Pafah1b subunits (lanes 2–4) or a control antibody (lane 5) did not co-precipitate the receptor. Blots were probed with antibodies against GFP to detect co-precipitated ApoER2 (upper panel) or total ApoER2 expression in the corresponding WCLs (middle panel). FLAG or HA antibodies were used to detect Pafah1b subunits (lower left panel) or Dab1 (lower right panel) in the corresponding WCLs. ApoER2 co-precipitated exclusively with Dab1. (D) Pafah1b2 and Pafah1b3 do not bind ApoER2 in a cell-free-system (TNT). In vitro translated proteins were radiolabeled with 35S and analyzed by SDS-PAGE (lanes 1–4). Equal amounts of ApoER2 were combined with the indicated proteins and immunoprecipitated with a control antibody (lane 5), or with antibodies against Dab1 (lane 6), Pafah1b2 (lane 7) or Pafah1b3 (lane 8). Proteins were detected by autoradiography. ApoER2 again co-precipitated only with Dab1. IP, immunoprecipitate; WCL, whole cell lysate.