Phosstop phosphatase inhibitor tablets

Cells (2 × 10⁶) were plated in six‐well plates and, after appropriate stimulations, they were lysed using radioimmunoprecipitation assay buffer (50 mmol L⁻¹ Tris–HCl, 150 mmol L⁻¹ NaCl, 1 mmol L⁻¹ ethylenediaminetetraacetic acid, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with cOmplete EDTA‐free protease inhibitor cocktail (Sigma) and PhosSTOP phosphatase inhibitor tablets (Sigma). Cell lysates (10 µg protein measured by bicinchoninic acid assay; Thermo Fisher Scientific, Waltham, MA, USA) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred on to nitrocellulose membranes (Bio‐Rad). Membranes were probed with antibodies (Supplementary table 2) and then developed by chemiluminescence using Clarity ECL (Bio‐Rad).

BV-2 cells were seeded into 6-well plates at 5.4 × 10⁶ cells per well and
incubated in 2 mL of DMEM (10% heat-inactivated FBS) for 18 hr. Then, the cells were
pretreated with each test agent under serum-free conditions for 1 hr and stimulated with
LPS (500 ng/mL) for 30 min. The cells were collected with a cell scraper (Techno Plastic
Products AG, Trasadingen, Switzerland) and centrifuged at 300 g for 10
min at 4°C, after which the supernatant was removed. Cells were resuspended in ice-cold
PBS and centrifuged at 300 g for 10 min at 4°C. To prepare the whole-cell
extract, the pellet was lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer
(Fujifilm Wako, Osaka, Japan) supplemented with cOmplete protease inhibitor cocktail
tablets (Sigma-Aldrich) and PhosStop phosphatase inhibitor tablets (Sigma-Aldrich). After
1 hr on ice, the extract solution was centrifuged at 16,260 g for 20 min
at 4°C, and the supernatant was collected. To prepare the cytoplasmic and nuclear
proteins, the pellet was lysed with protein extraction buffer, and then cytosolic and
nuclear proteins were extracted with an EzSubcell Extract kit (ATTO, Tokyo, Japan)
according to the manufacturer’s instructions. Total protein levels in the preparations
were determined with a Micro BCA Protein Assay kit (Thermo Fisher Scientific, Vernon
Hills, IL, USA).