Characterizing additional PITPβ interactions. Quantitative data are shown as mean ± SD, with the number of independent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, **P < 0.01, ns (non-significant) P > 0.05. (A) Co-precipitation experiment assessing the association of HA-tagged PLD2 with GFP-tagged PITPβ in cells, n = 5. A representative result is shown on the left and quantitation is shown on the right. (B) Co-precipitation experiment assessing the association of HA-tagged LPP3 with GFP-tagged PITPβ in cells, n = 5. (C) Dot blot analysis revealing that PITPβ binds directly to PC: PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; Chol, cholesterol; SM, sphingomyelin; DAG, diacylglycerol; PI(4)P, phosphatidylinositol 4-phosphate, n = 4. (D) Liposome binding study involving PITPβ incubated with liposome containing different lipids as indicated, n = 5. The pellet (P) fraction contains membrane-bound PITPβ, while the supernatant (S) fraction contains soluble PITPβ. (E) Liposome-binding study involving PITPβ incubated with Golgi-like liposomes, either having the full lipid composition or lacking a particular lipid as indicated, n = 3. The pellet (P) fraction contains membrane-bound PITPβ, while supernatant (S) fraction contains soluble PITPβ. A representative result is shown on the left and quantitation is shown on the right. (F) Dot blot analysis revealing that the catalytic mutant of PITPβ binds directly to PC, n = 2. (G) PITPβ can bind simultaneously to PA-containing liposomes (90% DOPC, 10% DOPA), coatomer, and soluble VAP-A (8-212). A representative result is shown on the left and quantitation is shown on the right, n = 3. (H) Proximity ligation assay examining the effect of siRNA against PLD2 on the proximity between PITPβ and ζ-COP, n = 4. Representative confocal images are shown on left, PLA signal (red), DAPI (blue). (I) Proximity ligation assay examining the effect of siRNA against LPP3 on the proximity between PITPβ and ζ-COP, n = 4. Representative confocal images are shown on left, PLA signal (red), DAPI (blue). Source data are available for this figure: SourceData F5.