Binding domains of Vps26 and SNX1 on Vps35. Vps35-binding activity of Vps26 (A) and SNX1 (B). At 48 h posttransfection with vectors expressing Vps26 or SNX1 and Flag-Vps35 wt or its deletion mutants, transfected 293T cell lysates were used for immunoprecipitation with anti-Vps26 or anti-SNX1 antibody, followed by Western blot analysis with anti-Vps26 or anti-SNX1 and anti-Flag (Vps35) antibodies. (A) Lanes 1 to 9 express Vps26, while lane 10 expresses only the vector. Vps35 construct expression was as follows: lanes 1 and 10, aa 1 to 796; lane 2, aa 1 to 500; lane 3, aa 1 to 307; lane 4, aa 1 to 230; lane 5, aa 1 to 120; lane 6, aa 120 to 307; lane 7, aa 120 to 796; lane 8, aa 500 to 796; and lane 9, aa 650 to 796. (B) Lane 1 expresses only the vector, while lanes 2 to 10 express SNX1. Vps35 construct expression was as follows: lanes 1 and 10, aa 1 to 796; lane 2, aa 1 to 500; lane 3, aa 1 to 307; lane 4, aa 1 to 230; lane 5, aa 1 to 120; lane 6, aa 120 to 307; lane 7, aa 120 to 796; lane 8, aa 500 to 796; and lane 9, aa 650 to 796.