Anti mouse and anti rabbit igg peroxidase conjugated antibodies

The monoclonal antibodies specific for α-Tubulin, PARP1 and the polyclonal antibody specific for ERβ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-AKT (pSer473) was from Cell Signaling Technology (Beverly, MA, USA), anti-mouse and anti-rabbit IgG peroxidase conjugated antibodies and chemical reagents were from Sigma-Aldrich (St Louis, MO, USA). ECL was from Amersham Pharmacia Biotech (Uppsala, Sweden). Nitrocellulose membranes and protein assay kit were from Bio-Rad (Hercules, CA, USA). Culture media, sera, antibiotics and LipofectAMINE transfection reagent were from Invitrogen (Carlsbad, CA, USA). The ERβ selective agonist KB9520 (see Additional file 1: Figure S1) was designed and synthesized by Karo Bio (Huddinge, Sweden). (KB9520 has been described previously [36 , 41 (link)–43 (link)]. The compound can be obtained following contact with Karo Bio AB [stefan.nilsson@karobio.se] and after signing of a Material Transfer Agreement together with a detailed protocol of planned study. A fee covering the cost of compound synthesis will be charged).

Hetero-dimer formation of the Hsp90 isoforms was assessed in yeast strains in which one Hsp90 isoform had a C-terminal GFP-tag and the other isoform had a C-terminal 6HA-tag. Tagging of the endogenous HSP90 isoforms was performed according to the protocol provided by Janke et al.^{45 (link)}. Cells were lysed in 50 mM Tris, pH 8, 20 mM NaCl and 0.15% NP40 and protease inhibitor Mix G (Serva). Co-immunoprecipitations were performed by incubating cleared lysates with GFP-Trap_A agarose (Chromotek) or Anti-HA agarose (Sigma) for 30 min. Beads were washes four times with lysis buffer and proteins were eluted by boiling the agarose for 5 min in 4× SDS buffer (NUPAGE). The following primary antibodies were used to probe the membrane: antiyeast Hsp90 polyclonal antibody (Pineda Antibody Service^{48 (link)} at a concentration of 1:30,000) anti-PGK1 monoclonal antibody (Novex, cat. no. 459250 at a concentration of 1:15,000), anti-HA monoclonal antibody (Sigma, product number H9658, at a concentration of 1:5000) and anti-GFP antibody (Roche, cat. no. 11814460001, at a concentration of 1:1000). The secondary antibodies were anti-mouse and anti-rabbit IgG-peroxidase conjugated antibodies (Sigma-Aldrich, cat. nos. A9044 and A0545, respectively, both used at a concentration of 1:20,000). Image acquisition was performed with an ImageQuant LAS4000 (GE Healthcare).

The monoclonal antibodies specific for α-Tubulin, E-Cadherin, AKT1, PARP1, HSC70, GADPH and acetylated-lysine and the polyclonal antibodies specific for ERβ, SIRT1, phospho-AKT (pSer473), AKT, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-AKT1 and AKT3 monoclonal antibodies were from Rockland Immunochemicals Inc. (Gilbertsville, PA, USA). Anti-mouse and anti-rabbit IgG peroxidase conjugated antibodies and chemical reagents were from Sigma-Aldrich (St Louis, MO, USA). ECL, nitrocellulose membranes and protein assay kit were from Bio-Rad (Hercules, CA, USA). Culture media, sera, antibiotics and LipofectAMINE transfection reagent were from Invitrogen (Carlsbad, CA, USA). The AKT-inhibitor MK-2206 was obtained from Selleck Chemicals (Houston, TX, USA). The, previously described ERβ selective agonist KB9520 [30 (link), 45 (link)] was designed and synthesized by Karo Bio AB (Huddinge, Sweden).