Phosphatase inhibitors cocktails 2 and 3

Cells were washed three times in ice-cold PBS and collected in PBS with 1% Triton X-100, 0.3% protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (cocktails 2 and 3, Sigma-Aldrich). Protein levels were quantified using Bradford reagent (Sigma-Aldrich). Equal concentration lysates in Laemmli sample buffer were boiled for 5 min; between 10 and 50 μg protein was resolved by SDS-PAGE and analysed via immunoblot using LI-COR 800CW and 680RD infrared secondary antibodies as indicated. Immunoblots were imaged and quantified using Odyssey software.

HEK293T cells were co-transfected with OSBP-GFP and VAPA-HA in combination with negative control siRNA or with siRNA against INPP5A or negative control siRNA. Where indicated, HEK293T cells were treated for 2 h with 50 μM IP3_AM. Cells were washed three times in PBS and lysed in immunoprecipitation (IP) buffer (20 mM HEPES, pH 7.4, 130 mM NaCl, 0.3% protease inhibitor cocktail (Sigma-Aldrich), 10 mM NaF, phosphatase inhibitors (cocktails 2 and 3, Sigma-Aldrich) and 1% NP-40). Samples were centrifuged at 10,000×g for 10 min at 4 °C, and the supernatant was then incubated with HA-Trap magnetic microparticles (ChromoTek) for 1 h at 4 °C. Beads were washed three times with IP buffer, and bound proteins were eluted in SDS–PAGE sample buffer, resolved by SDS–PAGE and analysed via immunoblot.