Phosphatase inhibitor cocktail

The S2 cells expressing the Torso proteins were sonicated, and the membrane fractions were prepared from the cell homogenate by ultracentrifugation at 103,000 × g, at 4 °C for 90 min, and dispersed in resuspension buffer (1:1 v/v solution of HEPES-buffered saline (HBS) and Schneider’s Drosophila medium). After an incubation with or without 10 nM BmPTTH for 10 min, the fractions were solubilized on ice for 15 min in resuspension buffer, containing 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 1 mM PMSF, and a Phosphatase Inhibitor Cocktail (Nacalai Tesque, Inc., Kyoto, Japan). The cell debris was removed by centrifugation at 20,000 × g, at 4 °C for 5 min, and the supernatant was subjected to immunoprecipitation with a monoclonal anti-FLAG antibody (mouse, #M185-3S, Lot. 002, Medical & Biological Laboratories Co., Ltd.) at 4 °C for 1 h, and with Protein A/G PLUS-Agarose (#sc-2003, Santa Cruz Biotechnology, Inc.) for an additional hour. The immunoprecipitates were washed three times with HBS containing 1% Triton X-100 and 0.5% sodium deoxycholate. The precipitated proteins were eluted with 150 μg/ml DYKDDDDK Peptide (Wako Pure Chemical Industries, Ltd.) and subjected to reducing SDS-PAGE. The autophosphorylation was determined by immunoblotting with an antibody that specifically recognizes phosphorylated tyrosine residues.

Cell pellets were lysed in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail (Nacalai Tesque, Inc.) and incubated for 30 min on ice. The supernatant was cleared via centrifugation at 15,000× g for 15 min at 5 °C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific), and equal amounts of protein (10 µg/lane) were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An ImageQuant LAS 3000 mini system (GE Healthcare UK Ltd., Buckinghamshire, UK) was used to detect the proteins. The following antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA): anti-PARP (9542S), anti-cleaved PARP (5625S), anti-phospho ERK1/2 (9101S), and anti-ERK1/2 (4696S). Anti-thymidylate synthase (TS) was produced in Taiho Pharm. Co., Ltd., (Tokyo, Japan), and anti-β-actin (clone AC-74) was purchased from Sigma-Aldrich.

Tissues were homogenized in RIPA buffer (10 mM Tris-HCl, pH8.0, 150 mM NaCl, 1 mM EDTA, 0.5% nonidet-40, 0.5% deoxycholate, 0.5% SDS, protease inhibitor cocktail, phosphatase inhibitor cocktail (Nacalai tesque) by Potter homogenizer and then sonicated on ice for 15 s by using a microtip sonicator. After centrifusion, protein concentration of supernatant was determined by bicinchoninic acid assay (Nacalai tesque). SDS sample buffer (x2∶125 mM Tris-HCl, pH 6.8, 4% SDS, 10% mercaptoethanol, 20% Glycerol, 0.002% bromophenol blue) was added into lysates. The membrane transfer was performed by semi-dry method. Western blot was performed as described previously [27] (link). Anti-Grp78 (BD bioscience), XBP1 (Abcam), GAPDH (Milipore) antibodies were used.

293 T cells constitutively expressing the Halo tag, Halo-UBAP2L or Halo-G3BP1 were lysed in lysis buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% NP-40) supplemented with a phosphatase inhibitor cocktail (Nacalai Tesque, Inc., Japan), protease inhibitor (Promega), 1.5 mM DTT, and RNase inhibitor (Takara) for 10 min on ice and treated with DNaseI(Takara) for 10 min at room temperature. The lysates were centrifuged at 15,000 rpm for 10 min and the supernatants were incubated with Halo Resin (Promega) for 12 h. The resin beads were washed with wash buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.005% NP-40) five times, and suspended in ML buffer (NucleoSpin miRNA: Takara) to isolate large and small RNAs.

HEK293T cells were transfected with DOCK11-BioFLAGHis or BioFLAGHis vector and T7-Ack1-His using Lipofectamine 2000 for 48 h. Cells treated with 10M-D42AN for 24 h were lysed with IP lysis buffer (Thermo) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Nacalai Tesque). Lysates were centrifuged at 4 °C for 15 min at 13,200 rpm, and the supernatant was applied to streptavidin-conjugated magnetic beads on a rotator for 3 h at 4 °C. The beads were washed with TBST and resuspended in NuPAGE LDS Sample Buffer (4×) containing 0.1 M DTT. The eluate was separated in a 3 to 8% SDS-PAGE and analyzed by Western blotting with HRP-conjugated mouse anti-FLAG-tagged monoclonal antibody or anti-Ack1 antibody.

The cells were washed in ice‐cold PBS and lysed using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma‐Aldrich, St. Louis, MO) and phosphatase inhibitor cocktail (Nacalai Tesque). For Western blot analysis, 10 μg total protein were loaded and run on 7.5–15% precast gradient polyacrylamide gels. The gels were transferred to nitrocellulose membranes at 20 V overnight. Membranes were blocked in 5% milk or Blocking One‐P (Nacalai Tesque) for 20 min and incubated with the primary antibody at 4°C overnight. The primary antibodies used for Western blot analysis are listed in Supporting Information Table S1. After washing, the membranes were incubated for 1 hr at room temperature with the secondary antibody. Specific protein bands were detected using the SuperSignal West Pico/Dura Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA).