Fig. 4

Hsp70-Bim PPI preferentially binds and stabilizes ERα36 instead of ERα66. A Western blot analysis of the relative levels of ERα36 and ERα66 using respectively specific monoclonal antibody in parental tamoxifen-sensitive MCF-7 and tamoxifen-resistant cell lines MCF-7/R1, MCF-7/R2 and MCF-7/TAM-R (left panel), as well as parental T47D and tamoxifen-resistant cell line T47D/TAM-R (right panel), using β-actin as a loading control. Bottom: relative level of ERα36 and ERα66. The data are expressed as mean ± SD (n = 3 biologically independent experiments) *P < 0.05, **P < 0.01 (one-way ANOVA test). B Co-IP analysis of Hsp70 interactions with ERα36 and Bim in tamoxifen-sensitive and resistant MCF-7 cell lines (left panel) and T47D cell lines (right panel). Bottom: relative level of protein in Hsp70 co-IP. The data are expressed as mean ± SD (n = 3 biologically independent experiments). *P < 0.05, **P < 0.01 (one-way ANOVA test). C Western blot of Hsp70 complexes using an H222 antibody raised against an epitope common to ERα36 and ERα66 in MCF-7/R1 extracts isolated by precipitation with a nonspecific IgG, Hsp70 antibody or Bim antibody. D Western blot analysis of the levels of ERα36 and Bim using specific monoclonal antibody in non-specific (NS) siRNA—and 2 independent human Bim-siRNA transfected MCF-7/TAM-R cells with vehicle control or S1g-2 (5 μM) treatment for 12 h. Right: the relative level of ERα36. The data are expressed as mean ± SD (n = 3 biologically independent experiments). **P < 0.01 (one-way ANOVA test); n.s. indicates no significance. E Western blot analysis of the levels of ERα36 in MCF-7/R1 upon treatment with vehicle control or MKT-077 (5 μM) for 12 h, using β-actin as a loading control