Figure 4.

Interaction of MuSK and NSF in mammalian cells. A, Identification of the domain in NSF to interact with MuSK. Various NSF constructs were generated by PCR and subcloned in pCMV with a Myc tag in the N terminus. N, Amino acids 1–211; D₁, amino acids 211–495; D₂, amino acids 496–752; D₁-D₂, amino acids 211–752; N-D₁, amino acids 1–495. The intracellular domain of MuSK (amino acids 515–868) was subcloned in pKH3 vector with an HA tag in the N terminus. HEK293 cells were cotransfected with indicated constructs, and 48 h after transfection, cells were lysed and subjected to immunoprecipitation (IP) with anti-HA antibody. Cell lysates were examined for the expression of NSF constructs (bottom panel). IB, Immunoblot. B, The kinase domain is sufficient for the interaction. Various MuSK constructs were generated by PCR and subcloned in pKH3 vector. HEK293 cells were cotransfected with indicated constructs, and cell lysates were subjected to immunoprecipitation with anti-Myc antibody.