Figure 2.

Verification of the interaction between the Hsp70 family and AR. A. Flag-Hsp70 or Flag-Hsc70 was transfected with GFP-NTD^AR into PC3 cells. In the lower panel, C4–2 cells were transfected with Flag-Hsp70 or Flag-Hsc70. Whole cell lysates (WCL) were prepared for co-IP using anti-GFP or Flag agarose beads as indicated. Flag vector was added to the transfection of Flag-Hsp70 group but not to the Flag-Hsc70 group. Less flag-Hsp70 vector was used due to the robust Flag-Hsp70 expression; flag vector was added such that equal amount of plasmids was added in each transfection reaction. B. GST and GST-Hsp70 purified from E. coli were used in the GST-pulldown of recombinant human androgen receptor. GST or GST-Hsp70 was mixed with human AR protein and then subjected to pulldown using glutathione-conjugated agarose. C. C4–2 cells were treated with 1 nM R1881 for 24 hours before fractionation. A total of 4 dishes were lysed. The nuclear extraction was obtained by adding 800 μL lysis buffer (175mM NaCl, 20 mM Tris-HCl, 1.5 mM MgCl₂, 0.5% NP-40, 15% glycerol, 2mM EDTA, pH7.5) to nuclear pellet of 4 dishes. Equal total protein amounts were used as input for both cytoplasmic (CE) and nuclear extract (NE).