Figure 4.

SNX13 is an ER-resident protein that associates with LDs via its C terminus. (A) Immunofluorescence microscopy of U2OS cells expressing SNX13-HA and VAPA-CFP. Proteins were detected with anti-HA antibodies or CFP fluorescence as indicated. Scale bar, 10 µm. (B) RPE cell expressing SNX13-GFP, treated with U18666A for 16 h. LAMP1 and BMP were detected using antibodies. Scale bar, 10 µm. (C and D) U2OS cells as in A, treated with oleic acid overnight. LDs were detected using LipidTOX, and cholesterol was detected using filipin. VAPA-CFP and SNX14-GFP were detected by intrinsic fluorescence. Shown in small boxes are enlargements of the boxed areas at left. Scale bars, 10 µm. (E) Cells as in A, visualized by confocal microscopy; green, SNX13; red, BMP; blue, LDs. Scale bars, 10 µm; enlarged inset and 3D rendering, 2 µm. (F) Schematic analysis of SNX13 constructs. (G–J) Localizations of constructs indicated in F. Cells were labeled as in E. Scale bars, 10 µm. Arrows indicate ring-like SNX13-ER domains in close contact with BMP-positive endolysosomes and LDs. PXA, PX-associated; RGS, regulator of G protein signaling; PX, phox homology.