1. Supplementary Figure S2 Ling Ren
K7M2
S Ins
Ezrin
pERM
ERM
Supplementary figure S2. Triton-X 100 fractionation of ezrin and phospho-ERM suggests that the majority of ezrin exists in the soluble (“dormant”) form in K7M2 osteosarcoma cells. To obtain the detergent-soluble and -insoluble fractions, cells were lysed in Triton X-100 lysis buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 1 mM EGTA, 3 mM MgCl2, 300 mM sucrose, 0.5 mM DTT, 0.5% Triton X-100) with protease inhibitors and 1 M Calyculin A. Following centrifugation (600 xg for 3 minutes) solubilized material was collected as the detergent-soluble fraction, expected to contain cytosolic “dormant” ezrin, and the pellet as the insoluble fraction, expected to contain actin bound “active ezrin”. Western analysis for ezrin, phospho-ERM and ERM of both fractions suggests that most ezrin (and ERM) is contained in the soluble fraction (“dormant ezrin”). The expression of almost all phospho-ERM in the insoluble fraction confirms the “active” state of ezrin found in this insoluble compartment.