Figure 4

Co-immunoprecipitation of neurexin-1β with acetylcholinesterase (AChE). A. Representative blots of co-immunoprecipitation of neurexin-1β with AChE from lysate of HEK293 cells transfected with neurexin-1β-1’-His (Nrxn-1β-1’, lacking alternatively spliced sequence 4 [SS4]) alone or AChE-S alone or both. Upper panel: Immunoprecipitation of AChE-S from the lysate of HEK293 cells transfected with both neurexin-1β-1’-His and AChE-S co-precipitated a large amount of 55-kDa neurexin-1β-1’ and a small amount of 58-kDa neurexin-1β-1’ but did not co-precipitate the O-glycosylated 73- and 91-kDa forms. The co-precipitated neurexin-1β-1’ was confirmed by blotting His. Lower panel: Immunoprecipitation of His co-precipitated AChE-S from the lysate of cells transfected with both neurexin-1β-1’-His and AChE-S. B. Immunoprecipitation of AChE-R co-precipitated a large amount of 55-kDa neurexin-1β-1’ and a small amount of 58-kDa of neurexin-1β-1’ from the lysate of cells transfected with both neurexin-1β-1’-His and AChE-R. C. In control experiments, absence of AChE antibody (lane 1) and application of IgG (lane 2) led to absence of co-precipitation of neurexin-1β-1’. Similarly, when the transfected cells were cultured in the presence of tunicamycin (Tunic.), no neurexin-1β-1’ was co-precipitated by anti-AChE (lane 4). D. Co-precipitation of neurexin-1β by anti-AChE from lysate of cells transfected with AChE-S combined with neurexin-1β-1’ (from left to right: lanes 1 and 2) or combined with neurexin-1β-3’ (with SS4) (lanes 3 and 4). The transfected cells were cultured in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 10 μM physostigmine (Physo.). Neurexin-1β was confirmed by blotting His. The presence of physostigmine increased co-precipitation of neurexin-1β by anti-AChE, and the amount of co-precipitated neurexin-1β-3’ was less than the amount of neurexin-1β-1’. E. Immunoprecipitation of AChE from the lysates of co-cultured HEK293 cells transfected with either Nrxn-1β-1’ or AChE-S before co-culturing. Anti-AChE co-precipitated the 55-kDa neurexin-1β-1’ from co-cultured the cells, and physostigmine increased co-precipitation of neurexin-1β-1’.