Figure 3
IFT88 is associated with COPII vesicles at the ER exit sites. (A) Representative immunofluorescence images of exogenously expressed GFP-IFT88, HA-DGKδ as well as endogenous calnexin in NIH3T3 cells. Arrows mark the positions of insets. MOC between IFT88 and DGKδ: 0.89 ± 0.065. (B) Schematic diagram and (C) a representative image of results from the proximity ligation assay showing that exogenously expressed FLAG-IFT88 and pEGFP-DGKδ are colocalized in the peri-nuclear region in NIH3T3 cells (the red signals adjacent came from another transfected cell). (D–F) Representative immunofluorescence images showing double staining of GFP-SEC16A and IFT88 (D), HA-DGKδ and GFP-SEC16A (E), as well as HA-DGKδ and SEC13 (F), respectively, in NIH3T3 cells. MOC: 0.68 ± 0.053 in (D), 0.81 ± 0.042 in (E), 0.63 ± 0.066 in (F). (G) IP-Western analysis of the interaction between exogenously expressed FLAG-IFT88 and GFP-SEC16A, (H) HA-DGKδ and GFP-SEC16A, as well as (I) HA-DGKδ and GFP-SEC13 in HEK293T cells. (J) Iodixanol density gradient sedimentation experiments in HEK293T cells, in which samples from 12 fractions were analyzed by Western blot for calnexin, IFT88, SEC13, and exogenously expressed HA-DGKδ. These experiments were reproduced in at least 3 independent experiments.
