Fig. 3
Hsp70-Bim PPI, instead of Hsp70-Bag3 PPI, confers increased level of tamoxifen resistance. Hsp70 from cell lysates was isolated through sequential immunoprecipitation steps using Bim antibody (A) or Bag3 antibody (B), followed by western blot using Hsp70 antibody. The data underneath the bands were calculated by the proportion of relative Hsp70 level pulled down by 5 × Bim or 5 × Bag3 antibody to that in cell lysates. The graphs in the right panel show (mean ± SD, n = 3 biologically independent experiments) relative levels of Hsp70-Bim PPI and Hsp70-Bag3 PPI. *P < 0.05, **P < 0.01 (one-way ANOVA test); n.s. indicates no significance. C Correlation of the relative levels of Hsp70-Bim PPI in different cell lines with the IC50 values of S1g-2. D Co-IP and western blot analysis of the relative levels of Hsp70-Bim PPI, Hsp70-Bag3 PPI and PARP cleavage in MCF-7/R1, MCF-7/R2 and MCF-7/TAM-R with a gradient of concentrations (0–10 μM) of S1g-2 treatment for 12 h (co-IP assay) and 24 h (PARP cleavage assay), respectively. E Correlation of the percentage of S1g-2-induced Hsp70-Bim PPI inhibition with the relative level of PARP cleavage. F Co-IP and western blot analysis of the relative levels of Hsp70-Bim PPI, Hsp70-Bag3 PPI and PARP cleavage in MCF-7/R1, MCF-7/R2 and MCF-7/TAM-R without or with 5 μM MKT-077 treatment for 12 h (co-IP assay) and 24 h (PARP cleavage assay), respectively. All figures represent the results from n = 3 biologically independent experiments