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. Author manuscript; available in PMC: 2011 Aug 12.
Published in final edited form as: Mol Cancer Res. 2009 Jun 30;7(7):1021–1032. doi: 10.1158/1541-7786.MCR-08-0582

Fig 3. Hsp90 inhibition facilitates EphA2 ubiquitination and degradation.

Fig 3

(A) (Left panel) HEK293 cells were transfected with 5 ug HA tagged EphA2 and treated for 6 h with 1 μM GA and/or 1 μM proteasomal inhibitor PS-341 for the last 3 h. Following lysis, 1 mg of total protein was immunoprecipitated with anti-HA antibody and ubiquitinylated protein was immunodetected. The blot was then stripped and reprobed with anti-HA antibody for analysis of total EphA2 levels. (Right panel) HEK293 cells were similarly transfected and treated with GA alone or with PS341 and EphA2 levels were dected from insoluble cell pellets following centrifugation, and from soluble protein lysate. B) Logarithmically growing PC3 cells were starved in methionine-free medium 1 h, pulse labeled for 1 h in the presence of 1 uM GA, and then chased for the indicated times in nonradioactive complete medium either lacking or containing 1 uM GA. Endogenous EphA2 was immunoprecipitated from equivalent protein and resultant gels were exposed to film and subject to densitometric analysis.