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. 2004 Jul;24(13):5667–5676. doi: 10.1128/MCB.24.13.5667-5676.2004

FIG. 3.

FIG. 3.

Active Lck mutants are dependent on Hsp90 for stability and ubiquitinated. (A) COS-7 cells were transfected with HA-tagged ubiquitin together with wild-type Lck (Lck) or the active mutant LckY505F (Y505F). Where indicated, cells were treated with GA for 90 min. Cell lysates were either subjected to immunoprecipitation (IP) with anti-Lck antibodies followed by blotting with anti-HA antibodies to detect ubiquitinated Lck (top) or were blotted for Lck directly (bottom). (B) COS-7 cells transfected with Lck, LckY505F (Y505F), or LckW97A (W97A) were treated with GA for 0, 1, 2, or 3 h. Cell lysates were blotted for Lck. The W97A mutation affects substrate binding of the SH3 domain. (C) COS-7 cells were transfected either with HA-ubiquitin on its own (−) or together with the indicated Lck constructs. Lck immunoprecipitates (IP) were immunoblotted with antiubiquitin antibodies to detect ubiquitinated Lck (upper gels), and cell lysates were blotted for Lck directly (lower gels). The positions of the 56-kDa, 59-kDa, and ubiquitinated form of Lck (Ub-Lck) as well as that of the immunoglobulin heavy chain (Ig HC) are indicated.