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. 2019 Feb 14;218(4):1335–1351. doi: 10.1083/jcb.201808133

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© 2019 Datta et al.

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Figure 2. — Snx14 is topologically anchored in the ER and interacts with LDs in trans. (A) LD flotation assay of OA-treated U2OS cells expressing Snx14^EGFP. Lanes indicate postnuclear supernatant (PNS), cytosol, total membrane, and LD float fractions. (B) Schematic diagram of Snx14 fragment constructs tagged with EGFP. Snx14^FL depicts the full-length human Snx14. Snx14^N is the N-terminal fragment from the start that includes PXA and RGS domains. Snx14^PXCN includes the PX domain and C-Nexin domains. Snx14^PX consists of PX and Snx14^CN represents C-Nexin domain. An AH in the C-Nexin domain is identified as depicted in the schematic diagram. Snx14^PXCNΔH indicates the PX and C-Nexin domain from which the AH is deleted. Snx14^FLΔH depicts the full-length Snx14 with AH deletion. (C) Western blot showing distribution of Snx14^FL, Snx14^N, and Snx14^PXCN tagged with 3XFlag among total membrane and LD fractions following OA treatment. (D) U2OS cells transfected with Snx14^FL, Snx14^N, Snx14^PXCN, Snx14^PX, Snx14^CN, Snx14^PXCNΔH, and Snx14^FLΔH, respectively, and treated with OA for 16 h. CoIF staining with α−EGFP (green) and α-HSP90B1 (ER marker, red) and LDs stained with MDH (blue) and imaged by confocal microscopy. Scale bar = 20 µm. Inset scale bar = 5 µm. Cartoons represent the localization of the respective Snx14 fragments with respect to ER and LD.