Mapping of the regions involved in the kinesin-1–Slp3 interaction. (A) Flag-Slp1, -Slp2, or -Slp3 and GFP-KLC1 were coexpressed into 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody and then separated by SDS-PAGE. Coprecipitated Slp proteins and KLC1 were immunoblotted with anti-Flag and anti-GFP antibodies. (B) For each Slp3 domain, a GFP fusion protein was cotransfected with HA-KLC1 into 293T cells. Cell lysates were immunoprecipitated with anti-GFP antibody and then separated by SDS-PAGE. Coprecipitated Slp3 domains and KLC1 were immunoblotted with anti-HA and anti-GFP antibodies. (C) GFP constructs of the N-terminal HPT or the C-terminal TRP were cotransfected with His-Slp3 in 293T cells. Cell lysates were immunoprecipitated with anti-GFP antibody. Coprecipitated Slp3 and KLC1 mutants were immunoblotted with anti-His and anti-GFP antibodies. (D) Cell lysates of a stable YT cell line expressing GFP-Slp3 construct were immunoprecipitated with an isotype control or with an anti-KLC1 antibody and immunoblotted with anti-KLC1 and anti-GFP antibodies. Blot results represent at least 3 independent experiments.