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

FIG. 5.

FIG. 5.

Phosphorylation of Ser42 and Ser59 is not required for Hsp90 monitoring and ubiquitination of Lck. (A) COS-7 cells transfected with Lck or Lck mutants were left untreated (−) or treated (+) with GA for 2 h, lysed, and blotted for Lck. Phosphorylation of active LckY505F (Y505F) on serine 59 is detected as a shift in molecular size from 56 to 59 kDa. Note the absence of the 59-kDa Lck form in Lck S59A/Y505F (S59A/Y505F). (B) COS-7 cells transfected with LckS42,59A/Y505F (S42,59A/Y505F) were treated with GA for 0, 1, 2, or 3 h. Cells were lysed and immunoblotted for Lck. (C) COS-7 cells were transfected with Lck constructs together with HA-ubiquitin. For the control (−), HA-ubiquitin was transfected on its own. Lck immunoprecipitates (IP) were immunoblotted with antiubiquitin (upper gel) or with anti-Lck antibodies (lower gel). 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.