Western blot was performed from lysates obtained using RIPA buffer supplemented with a protease inhibitor cocktail (Sigma).
PCR reactions were performed with 1 unit of Taq-polymerase (Life Technologies) in the manufacturers' buffer supplemented with 0.2 mM dNTP, in a total volume of 25 μl.
Cells were lysed using RIPA buffer supplemented with protease inhibitors.
The cellulase reaction mixture contained 0.5 μg of the total protein in the same buffer supplemented with a fluorescent substrate 4-methylumbelliferyl β-D-cellobioside, and incubated at 37°C for 6 h.
To prepare whole cell lysates, cell pellets were washed in PBS and lysed using standard RIPA buffer supplemented with a protease inhibitor cocktail (1∶50 dilution of lysis buffer).
Resting platelets were lysed with 1% Triton X-100 containing lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors.
Cells were stimulated with anti-CD3ε (clone HIT3a; 1 µg/mL, BD Biosciences, San Diego, CA)+anti-IgG2a (1 µg/mL, Southern Biotechnology Assoc., Birmingham, AL), as indicated, and lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Boehringer, Ridgefield, CT), 10 mM NaF, and 1 mM Na3VO4.
For protein extraction, neutrophils were lysed for 30 min at 4°C in NP-40 lysis buffer supplemented with a cocktail of protease inhibitors (2mM sodium orthovanadate, 1mM phenyl-methylsulfonylfluoride, 10 µg/ml leupeptin, 0.15 units/ml approtinin, 1 µg/ml pepstatin A) (Sigma) and centrifuged for 20 min to remove nuclei.
Briefly, B/TSM cells were lysed for 30 min 4°C in NP-40 lysis buffer supplemented with a cocktail of protease inhibitors (2 mM sodium orthovanadate, 1 mM phenyl-methylsulfonylfluoride, 10 µg/ml leupeptin, 0.15 units/ml approtinin, 1 µg/ml pepstatin A) (Sigma-Aldrich) and centrifuged for 20 min to remove nuclei.
Briefly, cells were lysed with RIPA buffer supplemented with a protease/phosphatase inhibitor cocktail.
