Further characterizing how PITPβ binds to coatomer and VAP-A. 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 < 0.05, ns P > 0.05. (A–D) Pulldown experiments using purified components to assess whether PITPβ interacts directly with coatomer (A), ARF1 (B), ARFGAP1 (C), and BARS (D), n = 5. (E) Pulldown experiment using purified components to identify the region in PITPβ critical for its direct interaction with coatomer, n = 3. A representative result is shown on the left and quantitation is shown on the right. (F) Pulldown experiment using purified components to identify a specific di-lysine sequence in truncated PITPβ critical for its direct interaction with coatomer, n = 8. The amino acid sequence of PITPβ (residues 137–204) is shown above, with di-lysine sequences highlighted in red. A representative result is shown below on left and quantitation is shown below on right. (G) MST binding curve measuring the interaction of coatomer with different PITPβ forms, WT PITPβ (magenta-colored curve) or K178A/K179A mutant (green-colored curve). The normalized fluorescence (Fnorm) is plotted against the logarithm of coatomer concentration. Each data point represents the mean ± SD of three independent experiments. The solid line represents the fit to a one-site binding model to yield a dissociation constant (Kd). (H) Pulldown experiment using purified components revealing that coatomer binding (tracked through β-COP) to the Wbp1 tail is not affected by PITPβ. Incubation involves 200 nM of each soluble component (coatomer and PITPβ forms). A representative result is shown on left and quantitation is shown on right, n = 4. Source data are available for this figure: SourceData FS3.