Functional sequelae of hsp90 inhibition. (A) Immunoblotting analyses of MM-1S cells treated with 17-AAG (500 nM for 0-24 hours) confirms that hsp90 inhibition suppresses the intracellular levels of multiple downstream effectors of the IGF-1R and IL-6R pathways, including the Raf-1 and IKK-α kinases (coupled with decreased constitutive phosphorylation of MEK1/2), the antiapoptotic proteins FLIP, XIAP, A1/bfl-1, cIAP2, and the pro-osteoclastogenic stimulator RANKL. In contrast, hsp90 inhibition by 17-AAG does not confer a significant effect on the intracellular levels of MEK1/2, for example. (B) Telomerase activity assay (by densitometric evaluation of the telomeric repeat amplification protocol (TRAP) method indicates that 17-AAG (500 nM, 0-24 hours) suppresses both constitutive and IGF-1 (200 ng/mL)-induced activation of the catalytic subunit of telomerase (hTERT). (C) 17-AAG treatment (750 nM for 0-36 hours) suppresses the NF-κB activity of MM-1S, as evidenced by NF-κB DNA binding enzyme-linked immunosorbent assay (ELISA). (D) 17-AAG treatment (750 nM for 0-36 hours) suppresses the activity of the 20S proteasome, as evidenced by 20S chymotryptic activity assay. (B-D) Error bars indicate SD.