Figure 2 - Overexpression, stabilization and depletion of HEF1.

(a) MCF-7 cells with tetracycline-repressed expression (MCF-7-tTA) of stably integrated HEF1 or GFP in the presence (+) or absence (-) of tetracycline, measured at 24 or 48 h after medium change. Western analysis with antibody to HEF1 demonstrates induction following tetracycline removal. -actin was used as a loading control. (b) Western blot analysis of MCF-7 cells infected by retroviruses expressing HA/thioredoxin-tagged peptide fusion proteins (HA–TRX). Levels of HEF1 are stabilized by specific HA–TRX peptides (P1-HEF1, P2-HEF1), but not by non-specific HA–TRX peptides (P1-NS), or by HA–TRX with no peptide inserted (NP). Antibody to HA shows comparable expression of HA–TRX fusions in all lanes. (c) Western blot analysis of MCF-7 cells treated with siRNAs to HEF1 (siHEF1 and siHEF1a) or a scrambled (Scr) or GFP (siGFP) oligonucleotide duplex shows efficient and specific HEF1 depletion at 48 and 72 h time points. The blot was stripped and re-probed with -actin as a loading control. All lanes shown were run on a single gel; white lines here and in the following figures indicate excision of intervening bands.