Mx 201

Blood was collected from the hearts of mice at the determined end points and centrifuged at 3000 rpm to isolate the plasma by a microvolume high-speed cooling centrifuge MX-201 (TOMY, Tokyo, Japan) and stored at −20 °C. The dorsal skin, livers, and kidneys were excised and homogenized with lysis buffer (Kurabo, Osaka, Japan), and then, the tissue lysate was centrifuged at 10,000 rpm. Plasma concentrations of interleukin (IL)-6, tumor necrosis factor (TNF)-α, creatinine, hyaluronic acid, endostatine, glutamate oxaloacetate transaminase (GOT), glutamic acid pyruvate transaminase (GPT), matrix metalloproteinase (MMP)-1, histamine, and angiopoitin 1 and 2 were measured using appropriate ELISA kits according to manufacturers’ instructions (IL-6, Enzo Life Sciences, Farmingdale, NY, USA; TNF-α, hyaluronic acid, and angiopoietin 2, R&D Systems, Minneapolis, MN, USA; MMP-1, MyBioSource, San Diego, CA, USA; histamine, Bertin Pharm, Montigny-le-Bretonneux, France; creatinine, Cayman, Ann Arbor, MI, USA; Endostatine, BioMedica, Vienna, Austria; angiopoietin 1, EIAAB Science, Wuhan, China; GOT and GPT, Wako, Osaka, Japan; ROS, Cell Biolabs Inc., San Diego, CA, USA). Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Cell extracts were prepared using an alkaline lysis method^{78 (link)}. 1 × 10⁸ cells from log-phase YE cultures were collected, washed with 1 ml H₂O, and suspended in 300 µl H₂O. After adding 300 µl of 0.6 M NaOH, the cell suspension was incubated at 30 °C for 5 min with the tube rotating. After centrifugation at 6000 rpm for 3 min (TOMY, MX-201), the alkali-treated cells were suspended with 140 µ of SDS sample buffer (60 mM Tris-HCl (pH6.8), 5% glycerol, 4% sodium dodecyl sulfate, 4% β-mercaptoethanol, 0.005% bromophenol blue) and incubated at 95 °C for 3 min. Cell extracts were recovered from the supernatant after centrifugation at 15,000 rpm for 1 min (TOMY, Kitman), separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (acrylamide to bisacrylamide ratio, 37.5:1), and transferred onto PolyScreen PVDF Transfer Membrane (Perkin Elmer, NEF1002001PK). To detect Chk1-HA, a mouse monoclonal antibody against the HA tag (16B12, Abcam, Cambridge, MA) (1:2000) and peroxidase AffiniPure goat anti-mouse IgG (heavy+light) (Jackson ImmunoResearch Laboratories, 115–035−146) (1:10,000) were used as the primary and secondary antibodies, respectively. The blots were developed using Supersignal West Femto substrate (ThermoScientific, 34095). Images were captured using ImageQuant LAS 500 (GE Healthcare).

Dorsal skin was isolated and homogenized in the lysis buffer (Kurabo, Osaka, Japan). The tissue extract was centrifuged at 10,000 rpm using a Tomy MX-201. The expressions of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF)-α were measured using PEG2 (Abcam) and TNF-α (Enzo Life Sciences, Farmingdale, NY, USA) ELISA kits, respectively, following the manufacturers’ instructions. Optical density was measured using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).