Further characterizing interactions of PITPβ. 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: ns (non-significant) P > 0.05. (A) Co-precipitation assay showing the association of GFP-tagged PITPβ mutant (FFAA) with HA-tagged PLD2, n = 4. Closed arrowhead indicates GFP-PITPβ. Open arrowhead indicates endogenous PITPβ. (B) Co-precipitation assay showing the association of GFP-tagged PITPβ mutant (K178A/K179A) with HA-tagged PLD2, n = 4. Closed arrowhead indicates GFP-PITPβ. Open arrowhead indicates endogenous PITPβ. (C) Golgi localization of wild-type and mutant forms of PITPβ as assessed by confocal microscopy. Representative confocal images are shown on left, PITPβ (green), giantin (magenta), bar = 10 μm. Quantitation of colocalization is shown on right, n = 3. (D) Western blotting showing the rescue levels of transfected PITPβ forms, n = 3. Closed arrowhead indicates GFP-PITPβ. Open arrowhead indicates endogenous PITPβ. (E) PITPβ binding to liposomes that contain increasing levels of PA, n = 3. A representative result is shown on left, and quantitation is shown on the right. (F) EM images showing PA-containing liposomes have similar size distribution after extrusion. Representative images were shown on the left, bar = 200 nm. Quantification of liposome size is shown on right, n = 3. (G) Cryo-EM images of PA-containing liposomes with inset showing intact membrane bilayers, bar = 100 nm, n = 3. (H) Highlighting regions in the PITPβ structure responsible for binding to coatomer (di-lysine motif), to VAP-A (FFAT motif), and to membrane (WW motif), as well as the catalytic domain (C94). (I) Efficacy of siRNA against PLD2 assessed by western blotting of cell lysate, with β-COP levels confirming similar levels of lysate examined, n = 4. (J) Efficacy of siRNA against LPP3 as assessed by western blotting of whole cell lysate, with β-COP levels confirming similar levels of lysate examined, n = 4. Source data are available for this figure: SourceData FS4.