Journal: The Journal of biological chemistry

Article Title: Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

doi: 10.1016/j.jbc.2023.105295

Figure Lengend Snippet: Figure 3. SEC22A associates with RAB18 and influences lipid droplet (LD) morphology. A, confocal micrograph to show overlapping localization of exogenous mEmerald-SEC22A (cyan) and mCherry-ER (red) in HeLa cells. Images are representative of at least 40 cells in three independent experiments. B, immunoprecipitation of exogenous HA-RAB18 from WT and RAB3GAP1-null HeLa cells. Cells were transfected with HA-RAB18 and/or mEmerald-SEC22A and lysed 24 h post-transfection. Anti-HA immunoprecipitates and input samples were subjected to SDS-PAGE and immunostaining for HA and GFP (mEmerald). C, confocal micrographs showing altered morphology in WT and RAB3GAP1-null HeLa cells coexpressing mEmerald-SEC22A and mCherry-RAB18; zoom shows colabeled vesicular structures. Images are representative of at least 10 cells in two independent experiments. D, RAB18 LFQ intensities from a reciprocal BioID experiment showing a reduced association between BioID2(Gly40Ser)-SEC22A and endogenous RAB18 in RAB3GAP-null compared with WT HeLa cells. Data were adjusted to account for nonspecific binding of RAB18 to beads and normalized by SEC22A LFQ intensities in each replicate experiment. Error bars represent SD. Data for other BioID2(Gly40Ser)-SEC22A-associated proteins are provided in Table S5. E, example of confocal mi- crographs and scatter plots to show effects of ZW10, NBAS, and SEC22A knockdowns on LD number and diameter. siRNA-treated IHH cells were loaded with 200 nM BSA-conjugated oleate, fixed and stained with BODIPY and DAPI, and imaged. Images were analyzed using ImageJ. Data are representative of three independent experiments. Two-tailed unpaired Welch’s t test #p < 0.05 and *p < 0.005. Bars represent 5 μm. BSA, bovine serum albumin; DAPI, 40,6- diamidino-2-phenylindole; ER, endoplasmic reticulum; HA, hemagglutinin; IHH, immortalized human hepatocyte; LFQ, label-free quantitation.

Article Snippet: Antibodies to ZW10, STX18, SPG20, RINT1, REEP4, BNIP1, C2CD2, TRIM13, WFS1, INPP5B, OSBPL2, and NBAS were obtained from Proteintech.

Techniques: Immunoprecipitation, Transfection, SDS Page, Immunostaining, Binding Assay, Staining, Two Tailed Test, Quantitation Assay