λ phosphatase buffer

Cells transfected with expression vectors for wt CREB-H FL protein were washed in ice-cold phosphate-buffered saline (PBS) and harvested by being scraped from the dishes, pelleted, and resuspended in λ-phosphatase buffer (New England Biolabs, Hitchin, United Kingdom) containing 0.4% NP-40, 0.5% Triton X-100, protease inhibitors (1× Complete Protease; Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF). As controls, two aliquots of the sample were treated with orthovanadate (10 mM) and sodium fluoride (20 mM) and incubated at 37°C either with or without added phosphatase for 1 h to reflect the starting material before incubation with phosphatase alone. Parallel samples were then incubated with 400 U of λ-phosphatase and incubated at 37°C for 1 h. The reactions were terminated by addition of SDS sample buffer to 1× and boiling for 5 min. Samples were analyzed by SDS–PAGE and Western blotting.

To a 50-µl lysate sample, from the 60-min α-factor arrest-release time point using Siz2-V5₃ cells, 200 µl of methanol, 50 µl of chloroform, and 150 µl of ddH₂O were added sequentially, followed by vortexing for 10 s after each addition. The final mixture was centrifuged for 2 min at 15,000 rpm. The resulting top layer was carefully removed, leaving the protein precipitate and the bottom layer. 300 µl of methanol was added to the remaining sample, and the mixture was vortexed for 10 s followed by centrifugation for 2 min at 15,000 rpm. Residual liquid was removed, and the resulting pellet was air-dried. The dried pellet was resuspended in 50 µl of 0.5% wt/vol SDS. To 10 µl of this sample, 10 µl of λ phosphatase buffer (New England Biolabs; B0761S), 10 µl of 10 mM MnCl₂, 1 µl λ phosphatase (New England Biolabs; P0753S), and 69 µl ddH₂O were added. For the -PPase sample, 1 µl λ phosphatase was replaced with 1 µl ddH₂O. Reactions were incubated at 30°C for 1 h, after which 20 µl of 50% TCA was added, and the samples were incubated overnight at 4°C. Samples were then centrifuged at 15,000 rpm for 20 min at 4°C, the supernatant was removed, and the remaining pellet was air-dried. The dried pellet was resuspended in 25 µl of 2× sample buffer and heated at 80°C for ∼15 min before Western blot analysis.