Figure 14. Afatinib nor ruxolitinib have ATPase inhibitory effects against HSP90 or HSP70.

(A) GBM12 cells were transfected with a plasmid to express HSP70-GFP or to express FLAG-tagged HSP90. Twenty four h after transfection chaperone proteins were immuno-precipitated using their tags. Equal portions of precipitate sepharose beads were immediately aliquoted into individual wells in a 96 well plate. Beads were resuspended in ATPase reaction buffer containing vehicle control; ruxolitinib; or afatinib (500 nM; 1000 nM; 1500 nM; 2 μM) in triplicate, and incubated for 30 min at 37°C. The reaction was started by addition of ATP-lite substrate. The plate was removed from the incubator and placed into a Vector 3 plate reader to determine the luminescence of the reactions under each treatment condition (n = 3 (× 3) +/− SEM). (B) GBM12 cells were treated with vehicle or [afatinib (1 μM) and ruxolitinib (1 μM)] for 6 h. Cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression levels of HSP70 and of HSP90 using antibodies raised to detect the NH₂-termini and COOH termini of the proteins.