FIGURE 1:
FAM21 directly interacts with retromer CSC. (A) Schematic representation of FAM21 constructs used. FAM21 contains 21 copies of the L-F-[D/E]3-10-L-F motif (LFa motif; black boxes). FN, FM, and FC contain repeats 1–6, 7–14, and 15–21, respectively. (B) GST-FAM21 pull-down of purified retromer CSC. Shown is a Coomassie blue–stained SDS–PAGE gel of input and bound samples for FN, FC, FM, and GST control. (C) GST-FM pull-down of retromer CSC with addition of increasing amounts of untagged FC protein. All input samples contained 1 nmol of GST-FM and 0.8 nmol of retromer CSC in the absence (lane 1) or presence of FC protein (lane 2, 1.5 nmol; lane 3, 10 nmol; lane 4, 20 nmol). Top, input samples separated by SDS–PAGE and stained with Coomassie blue. Bottom, diluted bound samples blotted against GST (loading control) and VPS29, respectively. (D) HeLa cells were transfected with shFAM21/HA-YFP-FAM21 rescue vectors expressing HA-YFP-FAM21 fusion proteins (amino acids 1–356 or R1-21), which were analyzed for association with VPS35 and WASH via immunoprecipitation.