FIG. 7.

(A) Highly specific antiphosphopeptide antibodies can distinguish between phosphorylated serines at positions 261 and 289. (B) In the GemS23A mutant that does not bind 14-3-3, phosphorylation of serine residue 289 is greatly reduced. Addition of calyculin A protects serine 289 from dephosphorylation. Lysates were prepared from Cos-7 cells transfected with wild-type Gem or Gem mutants. Phosphorylation of serines 261 and 289 was detected following immunoprecipitation with Gem monoclonal antibody P2D10 followed by Western blotting using antiphosphopeptide antibodies. Total Gem expression was determined using anti-Gem polyclonal antibody.