Phospho ezrin radixin moesin

IHC was performed on 5 μm–thick formalin-fixed paraffin embedded (FFPE) TMA or mouse tissue sections with cleaved-caspase 3 (Cell Signaling Technology, catalog no. 9661), phospho-Ezrin-Radixin-Moesin (Cell Signaling Technology, catalog no. 3726), or total ezrin (Millipore-Sigma, clone 3C12 catalog no. E8897) using the automated Ventana Discovery XT platform (Ventana Medical Systems) and Ethylenediaminetetraacetic Acid (EDTA) buffer for antigen retrieval (pH 8.0, 100°C). IHC-stained slides were scanned using ScanScope (Aperio Technologies) to obtain digital images. For mouse tissue sections, individual lung metastases were manually annotated and the percent positive cells within metastatic lesions for each marker was assessed by automated scoring with Halo (Indica Labs, Inc.) using a membrane/cytoplasmic-specific algorithm, for cleaved-caspase 3 or phosphorylation site in ezrin, radixin, and moesin (pTERM). Manual annotations also provided total number and mean size (in μm²) of metastatic lesions per tissue section. For TMA analysis, total ezrin was scored as described above.

Whole-cell lysates were prepared for SDS-PAGE as described previously (11 (link)). Briefly, 10–20 μg of protein were separated by SDS-PAGE, transferred to 0.45 μm polyvinylidene difluoride membranes (Bio-Rad), blocked with 5% nonfat dry milk in 1× TBS/0.1% Tween-20, and then probed with ezrin (Cell Signaling Technology, catalog no. 3145), radixin (Cell Signaling Technology, catalog no. C4G7), moesin (Cell Signaling Technology, clone Q480 catalog no. 3150), phospho-ezrin-radixin-moesin (Cell Signaling Technology, catalog no. 3726), phospho-Serine473 Akt (Cell Signaling Technology, catalog no. 9271), total Akt (Cell Signaling Technology, catalog no. 9272), phospho-Serine136 Bad (D25H8, Cell Signaling Technology, catalog no. 4366), total Bad (Cell Signaling Technology, catalog no. 9292), phospho-Serine536 p65 NFκB (Cell Signaling Technology, catalog no. 3031), total p65 NFκB (Cell Signaling Technology, catalog no. 8242), survivin (Cell Signaling Technology) or γ-tubulin (Sigma-Millipore, catalog no. T5326), with the appropriate secondary antibodies (Cell Signaling Technology).