Expression of Ezrin^WT in HS1 and HS2 spi-1 transgenic proerythroblasts. (A) Whole-cell lysates from HS1 preleukemic cells (749, 633, 812, 663, and 683) and HS2 leukemic cells (601, 606, 622, 931, and 980) were subjected to Western blot analysis using an antibody raised against ezrin. The blot was stripped and reprobed with an anti-actin antibody to visualize the equal loading of proteins. Molecular weight standards are indicated on the left of the panels. The membrane was exposed in an Imager, and the resulting signal was quantified using the ImageGauge software package (Fuji, Paris, France). Values were normalized to actin expression. Basal protein level was AU for 663 HS1 cells. The ezrin expression level for each cell line is indicated. Western blot is from a representative experiment. (B) Ezrin, radixin, moesin, and merlin expression. Whole-cell lysates from HS1 preleukemic cells (812 and 663) and HS2 leukemic cells (606 and 931) were subjected to Western blot analysis using antibodies raised against ezrin, radixin, moesin, merlin, and β-actin as a loading control. (C) Expression of ezrin in whole-cell lysates from one clone of 663 HS1 cells transfected either with pEF-neo or pEF-neo Ezrin^WT-VSVG constructs and cultured in the presence of Epo. Lysates were analyzed by Western blotting. The membrane was first probed with an anti-VSVG antibody and then with an anti-ezrin antibody. β-actin served as a loading control. Fold-increase in ezrin expression is indicated at the bottom of each lane. (D) Enforced expression of wild-type ezrin did not change the proliferation characteristics of transfected 663 HS1 cells (663H1-Ez^WT versus 663HS1-neo cells) whether cells were cultured in the presence (1 U/mL) or in the absence of Epo. Numbers of living cells were monitored at 24 and 48 hours using the Trypan blue (0.2% in PBS) exclusion staining (Sigma-Aldrich) and a Vi-Cell analyzer (Beckman Coulter, Villepinte, France). The mean number of living cells and standard deviations were determined from 3 experiments.