A role of Tex2 in the maintenance of lipid composition of LE/lys membranes. (A) Coomassie blue staining of purified 14xHis-Tex2-SMP. (B) In vitro lipid-binding assays for Tex2-SMP. Purified Tex2-SMP was incubated with NBD-tagged lipids and examined by native PAGE. Phospholipids, visualized by their fluorescence, comigrated with protein, visualized by Coomassie blue staining. (C) Ratio of fluorescence of Tex2-SMP-bound lipids to the protein level. Ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. Mean ± SD. (D) Schematic cartoon of non-targeting lipidomic analysis of endogenous GFP-Tex2. (E) Quantification of lipids bound to endogenous GFP-Tex2 from three independent assays. The lipid composition of total membranes in HEK293 cells are shown (Gao et al., 2022). (F) OptiPrep flotation assays showing the distribution of LE/lys membranes (anti-Lamp1) in consecutive 14 fractions from control or Tex2-KO HeLa cells with the eighth fraction being more abundant and specific. (G) Western blots showing the specificity of the fractions enriching ER membranes (anti-Calnexin) using the ER isolation kit from control or Tex2-KO HeLa cells, along with the purified LE/lys membranes from F. (H) Ratio of lipid species of the ER fractions or the LE/lys fractions in Tex2-KO relative to those from control HeLa cells from three independent assays. Mean ± SD. Source data are available for this figure: SourceData F9.