Fig. 1: SHIP164 interacts with RhoBTB3.

a Representative images of a live HEK293 cell expressing GFP-SHIP164 (green) and Halo-RhoBTB3 (magenta). Enlarged images of three boxed regions from the whole-cell image were shown on the bottom with line-scan analyses on the right. b Representative 3D rendering of a HEK293 cell expressing GFP-SHIP164 (green), Halo-RhoBTB3 (magenta), and Golgi-BFP (TM of B4GALT1) (blue) with y–z projection to the right and x–z projection to the bottom. c GFP-Trap assays demonstrate an interaction between GFP-SHIP164 and Halo-RhoBTB3 in HEK293 cells. d Co-IP assays show interactions between endogenous SHIP164 and endogenous RhoBTB3 or Vps26B in HEK293 cells. e Co-IP assays show interactions of endogenous SHIP164 and endogenous Vps26B with GFP-RhoBTB3 in HEK293 cells. f Schematic cartoon of RFPnb-mediated recruitment of BFP-RFPnb-RHOBTB3 to lipid droplet membranes (ACSL3-mCh) in the presence of GFP-SHIP164. g Representative images of a HEK293 cell expressing BFP-RFPnb-RhoBTB3 (blue), GFP-SHIP164 (green) and ACSL3-mCh (magenta) with insets. h Pearson’s correlation coefficient of GFP-SHIP164 vs ACSL3-mCh either in the presence (22 cells) or the absence (25 cells) of BFP-RFPnb-RHOBTB3. n > 3 independent experiments. Two-tailed unpaired Student’s t-test. Data are represented as mean ± SD. i Representative images of a fixed HEK293 cell stained with pre-cleared SHIP164 antibody and RhoBTB3 antibody (magenta) with two insets on the bottom. j Pearson’s correlation coefficient of endogenous SHIP164 puncta and RhoBTB3 (19 cells). Two-tailed unpaired Student’s t-test. Data are represented as mean ± SD. k Representative images of a fixed HEK293 cell expressing TGN46-Halo (blue) stained with pre-cleared SHIP164 antibody (green) and EEA1 antibody (magenta) with two insets on the bottom. l The distribution of endogenous SHIP164 puncta relative to TGN–EE junctions. 501 SHIP164 puncta and 743 potential TGN-EE junctions from 13 cells. Data are represented as mean ± SD. m Representative images of a HEK293 cell expressing YFP-EEA1 (magenta) and Halo-RhoBTB3 (green). Left: whole cell images; right: time-lapse images showing dynamic interactions between EEs and RhoBTB3-labled Golgi vesicles (Supplementary Videos S1, S2: time interval: 8 s). n Duration of the contacts between EEA1-labeled EEs and RhoBTB3 Golgi vesicles from 3 independent assays. o Representative images of a HEK293 cell expressing ER-tagRFP (magenta) and Halo-RhoBTB3 (green) (Supplementary Video S3, time interval: 14 s). p Duration of the contacts between the ER and RhoBTB3 Golgi vesicles from 3 independent assays. q GST-pull-down assays demonstrate that purified Chorein N ___domain of SHIP164, but not GST tag, was pelleted with purified His-RhoBTB3 in vitro. The Coomassie blue staining of proteins used in the assay was shown on the right. Red arrows denote the purified proteins in the gel. Scale bars: 10 μm in the whole cell images and 2 μm in the insets (a, b, g, i, k, m, o). Time shows in seconds.