Radixin

The antibodies against the following proteins were used: human ERα (Santa Cruz Biotechnology, #sc-543); HA (hybridoma clone 12CA5; kindly provided by Peter Angel, DKFZ, Germany); Rb (BD Biosciences, #554136); c-Jun (Cell Signaling Technology, #9165), phospho-c-Jun (Ser63) (Cell Signaling Technology, #9261), myc Tag (Upstate, #06-549); E1A (Calbiochem, #DP11-100UG; Santa Cruz Biotechnology, #sc-58658); RhoA (Cytoskeleton, #ARH03), Rac1 (Upstate, #05-389); Cdc42 (Thermo Scientific, #89857D), Ezrin (Cell Signaling Technology, #3145), Radixin (Cell Signaling Technology, #2636), Moesin (Cell Signaling Technology, #3150).

Cells were lysed as previously described [27 (link)]. Protein concentration was estimated using the Pierce^TM BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Protein lysates were separated by SDS-PAGE and subjected to Western blotting using the indicated primary antibodies (ezrin, radixin, moesin, phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558), cleaved caspase-3, and β-actin, 1 : 1000, Cell Signaling, Danvers, MA). HRP-linked goat α-rabbit IgG secondary antibody (1 : 10 000; Jackson ImmunoResearch, West Grove, PA, USA) and Amersham^TM ECL^TM Western blot detection reagents (GE Healthcare, Madison, WI, USA) were used to visualize protein bands. Relative band intensity was quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/).

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).