Fig. 4. Interaction between SKD1(E235Q) and NPC1. (A) Schematic representation of expressed SKD1 proteins. Each tag is shown in gray. (B) Flag-NPC1 ubiquitylation. COS cells were transfected with the Flag-NPC1 construct together with an empty vector or His₆-SKD1 constructs. 48 hours after transfection, anti-Flag immunoprecipitation (IP) products were subjected to immunoblotting (IB) with anti-Flag or anti-ubiquitin (P4D1). Molecular weights are given on the left (kDa); the arrowhead indicates the top of the separating gel. (C) Intracellular localization of GFP-SKD1 and Flag-NPC1. COS cells expressing Flag-NPC1 and GFP-SKD1 wild-type (wt; upper) or the E235Q mutant (EQ; lower) were fixed and stained with anti-Flag antibody. Bound antibody was visualized with Alexa Fluor 546-conjugated secondary antibody. Results shown are the representative images obtained with a confocal microscope. (D) Co-precipitation of Flag-NPC1 and GFP-SKD1 proteins. 0.5% CHAPS extracts were prepared from COS cells transfected with Flag-NPC1 together with GFP-SKD1wt or E235Q constructs. Anti-Flag immunoprecipitation products were analyzed by immunoblotting with the indicated antibodies. Cell extracts (IN: input) were loaded on lanes 1, 3, 5 and 7, and corresponding immunoprecipitation products were loaded on lanes 2, 4, 6 and 8. (E) Co-purification of endogenous NPC1 in CHO cells with His₆-SKD1 proteins. Upper; intracellular localization of GFP-SKD1. Results shown are the representative images obtained with a confocal microscope. Lower; affinity purification. 0.5% CHAPS extracts were prepared from cells transfected with an empty vector, His₆-SKD1wt or E235Q constructs. His₆-tagged proteins were affinity purified with metal affinity resin and were analyzed by immunoblotting with the indicated antibodies. Cell extracts (IN; input) were loaded on lanes 1, 3 and 5, and corresponding affinity-purification products (AP) were loaded on lanes 2, 4 and 6. (F) In vitro interaction between GST-SKD1 and Flag-NPC1. GST-fused proteins, as shown in the schematic representations, were expressed in E. coli and immobilized on glutathione sepharose (left, CBB: Coomassie Blue staining). 0.5% CHAPS extracts (input) were prepared from COS cells expressing Flag-NPC1. Cell extracts were incubated with immobilized GST-fused proteins in the absence or presence of 0.5 mM ADP, ATP or ATPs. Bound proteins were eluted with glutathione and analyzed by anti-Flag immunoblotting (right). Molecular weights are given on the left (kDa). All results shown are representative and were reproduced at least twice.