Cellular Fractionation Protocol for Protein Analysis

Cellular fractionation was performed as described elsewhere (Suzuki et al, 2010 (link)). Briefly, cells growing on the P100 dish were washed with ice-cold PBS, scraped, and collected in 1.5-ml micro-centrifuge tube. After centrifugation (10 s, 1.7 × 10³ g), the pellet was resuspended in 900 μl of ice-cold 0.1% NP40 (IGEPAL CA-630, I8896; Sigma-Aldrich) in PBS, and 300 μl of the lysate (whole cell lysate fraction, W) was transferred to a separate tube. The remaining material was centrifuged (10 s, 1.2 × 10⁴ g), and the pellet was resuspended in 1 ml of ice-cold 0.1% NP40 in PBS and centrifuged (10 s, 1.2 × 10⁴ g). The pellet (∼20 μl) was resuspended in 180 μl of 1 × Laemmli sample buffer (nuclear fraction, N). Lysates were sonicated and boiled for 1 min at 95°C.