Sarm1 interacts with the cytoplasmic domain of Sdc2. (A) GST–Sdc2 pull-down assay with extract prepared from P21 mouse brain and immunoblotted (IB) with Sarm1 antibody. GST alone was used as a control. (B) Co-immunoprecipitation of Sdc2 and Sarm1 from mouse brain. Sdc2 antibodies and nonimmune rabbit IgG were used in the immunoprecipitation. The precipitates were immunoblotted with Sarm1 and Sdc2 antibodies as indicated. The arrowhead indicates the position of Sarm1. Because of the heterogeneity of glycosylation, SDS-PAGE resulted in a ladder of Sdc2-immunoreactive bands. (C) Schematic of Sdc2 constructs. TM, transmembrane domain; C1, conserved region 1; V, variable region; C2, conserved region 2; Myr, myristoylation modification. (D) Sdc2 directly interacts with Sarm1. Sdc2-GST fusion proteins were used to pull down purified MBP-Sarm1 fusion protein in the presence of glutathione agarose. The presence of Sarm1 in the precipitate was examined by immunoblotting with MBP antibody. The relative amounts of GST fusions used in the pull-down assay are indicated by Coomassie blue staining. Sdc2 forms an SDS-resistant dimer through its transmembrane domain. The Sdc2 dimer and monomer are marked by the arrowhead and arrows, respectively. (E) The cytoplasmic domain of Sdc2 is sufficient for the interaction with Sarm1. HEK293T cells were cotransfected with HA-G-S2C, Myc-tagged Sarm1, and vector control, as indicated. Immunoprecipitation was performed using a Myc-tag antibody. The precipitates were then analyzed by immunoblotting with Myc and HA antibodies. The positions of HA-G-S2C and immunoglobulin light chain (IgL) are indicated. (F) Three regions (C1, V, and C2) are involved in the interaction with Sarm1. Various GST–Sdc2 fusion proteins were used to pull down Sarm1 from mouse extract. The results were immunoblotted with Sarm1 antibody. Coomassie blue stain indicated the relative amounts of GST fusions used in the pull-down assay. Molecular mass standards (kD) are indicated next to the gel blots.