Ef0652

For immunoblot assay, the seedlings of pHsfB2b::HsfB2b-eGFP were harvested at each time point. Total proteins were prepared from 100 mg of harvested samples in protein extraction buffer (50 mM Tris–Cl pH 7.5, 150 mM NaCl, 10 mM MgCl₂, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM 1,4-Dithiothreitol (DTT), 1Χ complete Mini, and EDTA-free protease inhibitor cocktail (Roche). Total proteins were separated by sodium-dodecyl sulfate (SDS)-PAGE. For phosphatase assay, total proteins were treated with or without alkaline phosphatase (Thermo scientific, EF0652) for 1 h at 37 °C, then separated by SDS-PAGE. The proteins were transferred to PVDF membranes (Amersham Biosciences) and probed with anti-GFP (Clontech, JL-8, 1:10,000 dilution) or anti-myc (Santa Cruz Biotechnology, sc-40, 1:10,000 dilution) antibodies overnight at 4 ℃. The samples were then probed with horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling, #7076, 1:10,000 dilution) antibodies at room temperature. The signals were detected using ImageQuant LAS 4000 (GE Healthcare) with WesternBrightTM Sirius ECL solution (Advansta).

HeLa cells were transfected with pRK5-HA-RPTOR (Addgene plasmid 8513,⁵² (link) gift from David Sabatini) 48 h before the experiment. HA-RPTOR was immunopurified using an HA antibody. A control (mock) IP was performed with rat IgG. The immunoprecipitates were dephosphorylated with alkaline phosphatase (10 U; Thermo Scientific, EF0652) for 1 h at 37°C, and washed with IP lysis buffer, followed by a washing step with kinase assay buffer (20 mM HEPES, pH 7,4, 150 mM KCl, 10 mM MgCl₂). Recombinant PLK1 (0.1 µg; Enzo Life Sciences, BML-SE466–0005) was added to RPTOR and mock IPs as indicated. The kinase-substrate mixture was preincubated on ice for 15 min with BI2536 (100 nM), Torin1 (250 nM) or carrier, respectively. An ATP mix with 1 mM cold ATP (GE Healthcare, 27–1006–01) and 5 to 10 µCi [γ-33P] ATP (PerkinElmer, NEG302H250UC) was added and incubated for 30 min at 30°C with gentle shaking. Samples were washed once with kinase assay buffer before resuspension in 1x SDS sample buffer and heated for 15 min at 68°C. Samples were separated by SDS-PAGE and phosphorylation was analyzed by autoradiography. For quantification the signal that was measured for the condition without PLK1 was considered as background and thus subtracted. For nonradioactive assay the same protocol was performed, 0.4 µM cold ATP was added and samples were analyzed by immunoblotting.