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. 2000 Jul 10;150(1):193–204. doi: 10.1083/jcb.150.1.193

Figure 2.

Figure 2

T567D ezrin and endogenous phosphorylated ERM proteins are preferentially monomeric at the plasma membrane. A, Membrane or cytosolic extracts from P, E, A, and D cells were resolved by gel filtration chromatography on a superose-6 column. Fractions 15–41 were analyzed by SDS-PAGE and immunoblotted with antiezrin antibodies (P) or anti-VSV G antibodies (E, A, and D). T567D ezrin exhibited a strongly reduced amount of oligomers at the membrane, but not in the cytosol. B, Total, cytosolic, and membrane extracts were immunoblotted with 297S mAb, recognizing all three ERM proteins when phosphorylated on this conserved threonine (pERM), or with a mixture of antibodies specific for ERM. Ezrin and radixin comigrated at ∼80 kD, and moesin migrated at 75 kD. Phosphorylated ERM proteins were strongly enriched in the membrane fraction. C, LLC-PK1 cells were pretreated with calyculinA, a protein phosphatase inhibitor, and the membrane extract was resolved by gel filtration chromatography. Oligomeric and monomeric fractions were pooled, and immunoblotted as in B. Monomers were preferentially phosphorylated over oligomers.