Fig. 1: Characterization of purified human HSL.

a The linear diagram of human HSL. b Size exclusion chromatography (SEC) of human HSL with a C-terminal FLAG tag. The FLAG-tagged HSL transiently expressed in Expi293F cells was firstly purified by anti-FLAG tag affinity resins, then further purified by an SEC column (Superdex 200 Increase). The peak fractions were analyzed by SDS-PAGE and visualized by Coomassie blue staining. The asterisk indicates a contaminant that was supposed to be a chaperone. We purified HSL several times, and the data in b represent the results of one such purification. c, d The enzymatic activity of the purified HSL was evaluated using pNPB (c) or the EnzChek lipase substrate (d) as the substrate of HSL, respectively (see methods). e Illustration of artificial lipid droplets (ALDs) preparation. ALDs were prepared by mixing phospholipids (DOPC/DOPE) with neutral lipids triacylglycerol (TAG). Created in BioRender. Hu, Q. (2025) https://BioRender.com/t74d619. f Illustration of the flotation assay. HSL or the control protein MBP was incubated with ALDs for 1 h at room temperature, and then separated by sucrose gradient centrifugation. After centrifugation, the mixture was divided into three fractions, including the bottom (280 µL), middle (260 µL), and top (100 µL) fractions. The ALDs were in the top fraction. g Evaluation of the proteins in the three fractions from the sucrose gradient centrifugation by SDS-PAGE followed by Coomassie blue staining. T, M, and B indicate the Top, middle, and bottom fractions, respectively. For each fraction, 20 μL of the sample was loaded on the SDS-PAGE gel. The gels in g are the results of a representative experiment out of three independent experiments. The asterisk indicates a contaminant that was supposed to be HSP70. h Quantification of the percentages of HSL and MBP in the top fractions. HSL or MBP was incubated with the assay buffer (no ALDs), or with ALDs prepared with DOPC (100%) or a mixture of DOPC (75%) and DOPE (25%), and then separated by sucrose gradient centrifugation. The data in c, d, and h represent the mean ± SD of three independent measurements. Source data are provided as a Source Data_Fig. 1 file.