Determination of detergent-resistant components of NIH3T3 cells before (C) and after treatment with kinase, staurosporine (ST), or phosphatase inhibitors, calyculin A (CA) or pervanadate (PV). The subconfluent cells were rinsed with PBS(+), and detached by treating with 0.02% EDTA in PBS. The suspended cells were incubated with or without 1 μM calyculin A, 1 μM staurosporine, or 100 μM pervanadate in PBS(+) for 10 min at 37°C. The cells were extracted for 5 min on ice with an equal volume of 2× detergent extraction buffer containing 2% of Triton X-100 or DOTMAC. The lysates were centrifuged at 15,800 × g for 5 min at 4°C. The resulting pellets were solubilized in SDS sample buffer and subjected to SDS-PAGE on a 9% polyacrylamide gel electrophoresed. Polypeptides were stained with Coomassie Brilliant blue (CBB). β-Actin, moesin, and threonine558-phospho-moesin were detected by immunoblot analysis.