Figure 3

GNMT interacts and colocalizes with NPC2 in cytosol. 293T cells were cotransfected with GNMT-Flag and NPC2-HA for 24 h and harvested for coimmunoprecipitation. (A) GNMT was precipitated by anti-Flag beads and immunoblotted with anti-HA to detect NPC2 expression (arrowhead). NPC2 was precipitated by anti-HA beads and immunoblotted with anti-Flag to detect GNMT expression (arrowhead). (B) SK-Hep1 cells were transfected with GNMT-Flag for 24 h and harvested for coimmunoprecipitation. GNMT was precipitated by anti-Flag beads and immunoblotted with anti-NPC2 antibody to detect endogenous NPC2 expression (arrowhead). (C) Liver lysates from WT mice were precipitated with anti-GNMT antibodies and immunoblotted with anti-NPC2 antibodies to detect endogenous NPC2 expression (arrowhead). (D) Cytosol and lysosome fractions were isolated from the liver tissues of WT mice. Western blot analysis was used to detect subcellular NPC2 and GNMT localization. LAMP1 and cathepsin D are indicated as lysosome markers; α-tubulin is indicated as a cytosol marker. (E) Liver cytosol fractions from WT mice were precipitated with anti-GNMT antibodies and immunoblotted with anti-NPC2 antibodies to detect endogenous NPC2 expression (arrowhead). (F) HuH7 cells were transfected with GNMT-Flag and fixed for immunofluorescence assays using antibodies against Flag, NPC2 and Lysotracker to detect colocalization among GNMT, NPC2 and lysosomes. Colocalization percentage is shown in each merge panel. Scale bar, 20 µm. IB, immunoblot.