Tissues were homogenized in RIPA buffer and lysed for 30 min on ice.
After stress exposures or anti-Fas antibody treatment, the cells were washed twice with ice-cold PBS buffer, and lysed for 1 h at 4 °C in 1 ml of 20 mM Tris HCl (pH 7.5) containing 1% Nonidet P-40, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate, and 1 × proteinase inhibitor cocktail.
TL-HMECs expressing HA/FLAG-PVRL4 or HA/FLAG-GFP were gently detached off the tissue culture surface with enzyme-free cell dissociation buffer, washed, and lysed for 30 min in MCLB lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40) in the presence of protease and phosphatase inhibitors (Roche), followed by centrifugation at 14,000 rpm at 4°C.
Subsequently, cells were washed with phosphate buffer saline and lysed for 10 min at 4 °C with lysis buffer comprising 50 mM Tris HCl (pH 7.4), 150 mM NaCl, 1 % Triton-X-100, Protease- and Phosphatase inhibitors (Thermo Scientific).
The nuclear P1 fraction was washed once in buffer A and lysed for 30 min on ice in 250 µl buffer B (3 mM EGTA; 0.2 mM EDTA; 1 mM DTT; protease inhibitor cocktail).
In all, 3 × 10 cells were resuspended in 200 μl radio-immune precipitation assay (RIPA) buffer (Roche) and lysed for 15 min at 4 °C.
Cells were harvested using Cell Dissociation Buffer (Sigma) and lysed for 30 min at 4°C in PBS, 1% Nonidet P-40 substitute, 50 mM Tris (pH 7.5), 5 mM EDTA, 150 mM NaCl, protease inhibitor mixture (Roche Diagnostics), and 5 mM Iodoacetamide.
The pellets were resuspended in RBC Lysis buffer (eBioscience, San Diego, CA) and lysed for 5 min after which the samples were spun down for 5 min at 1,500 rpm.
Cells were then scraped into 300 μl of lysis buffer (10 m M Tris pH 7.5, 150 m M NaCl, 0.25% NP40, with one protease inhibitor tablet (Roche, Welwyn Garden City, UK) per 10.0 ml of buffer), frozen, thawed, and lysed for 30 min at 4 °C.
The next day, the cells were stimulated with agonists in 50 µl of 1× stimulation buffer for 30 min at 37°C and lysed for 10 min with 9 µl of lysis/detection buffer.
For brain lysis, a 10% homogenate was prepared in Ripa buffer with protease inhibitors (Complete; Roche), and lysed for 1 h on ice.
