A2095

Cells were grown to a density of 2 × 10⁷ cells/ml in 2 l of SD + CSM – URA + 134 μM methionine and then pelleted, washed with water, and resuspended for 1 h in 2 l of SD + CSM – URA – methionine. Cells were washed with IP buffer (10% glycerol, 0.75% NP-40, 150 mM NaCl, 1.5 mM magnesium acetate, 50 mM Tris, pH 7.8) plus protease inhibitors (04693116001; Roche) and then pelleted and frozen in liquid nitrogen. Cells were lysed with a Spex SamplePrep Freezer/Mill 6870 (Spex, Metuchen, NJ), after which the ground cell material was resuspended in IP buffer plus protease inhibitors + phosphatase inhibitors (04906837001; Roche). Nonsoluble material was removed by centrifugation, and Met30-3xHA was precipitated from the soluble material by incubation for 75 min at 4°C with 120 μl of anti-HA conjugated beads (A2095; Sigma-Aldrich) that had been prewashed with IP buffer plus protease and phosphatase inhibitors. Unbound proteins were removed by repeated washing with IP buffer plus protease and phosphatase inhibitors, after which bound protein was eluted by treating beads twice with 0.5 ml 500 mM ammonium hydroxide and 0.5 mM EDTA for 20 min at room temperature.

To generate the ProPYLs:PYLs-HA-YFP constructs, the promoter fragments of PYLs was cloned into the SalI and BamHI sites of the modified pSAT vector with YFP and 3HA tags at the C terminus. The coding region of PYLs was cloned between the PYL promoter and the YFP sequence. The whole insertion cassette was digested with PI-Psp1 and inserted into pRCS2-htp binary plasmids. These plasmids were transformed into pyr1pyl1/2/3. Transgenic plants were verified by western blot assay.
The tandem affinity purification was performed as previously described (Zhao et al., 2016 (link)). The transgenic Arabidopsis plants expressing ProPYLs:PYLs-HA-YFP were used for tandem affinity purification (TAP). Ten-day-old seedlings (3–4 g fresh weight) were soaked for 30 min with or without 0.8 M mannitol. Generally, proteins were pre-purified with monoclonal anti-HA agarose with antibody produced in mouse (A2095, Sigma) in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 x protease cocktail, Roche), and eluted by HA peptide (Abcam, ab13835) with a concentration of 0.5 μg/μL in 200 μL IP buffer. PYLs-HA-YFP and their interacting proteins were then affinity purified with GFP-Trap Agarose (Chromotek, gta-20) and identified by mass spectrometric (MS) analyses. All steps were performed at 4°C.
The sequence of the peptides is listed in Table S3.

Cultured cells were collected by centrifugation, washed twice in PBS and lysed on ice in lysis buffer (0.5% Triton-X100, 150 mM NaCl, 1 mM EDTA, 20 mM TRIS-HCl pH 8) containing complete protease and phosphatase inhibitor cocktails (Sigma P8340 and Roche 04906845001, respectively), spun for 10 min at 10,000 g in an Eppendorf 5430R at 4 °C, followed by the addition of 20 µl anti-HA or anti-FLAG slurry (Sigma A2095 and A2220, respectively) to the cleared supernatant fraction. After incubation at 4 °C for 2 h, beads were collected by centrifugation at 5,000 g for 30 s at 4 °C followed by extensive washes in lysis buffer, and finally boiled in 20 µl 2× Laemmli sample buffer (Sigma S3401).