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. 2013 Oct 3;288(46):33146–33155. doi: 10.1074/jbc.M113.504407

FIGURE 2.

FIGURE 2.

Tyrosine 262 is the major phosphorylated tyrosine in Human GADD34. Panel A, cells expressing control vector (C), WT GADD34-FLAG (GADD34), or the mutant GADD34 (Y262F)-FLAG (Y262F) plasmids were subjected to immunoprecipitation using FLAG-M2 beads. The bound proteins were eluted as described in the methods and expression of GADD34 and its phosphotyrosine content analyzed by immunoblotting. Representative ratios of phosphotyrosine (p-Tyr) content relative to the total GADD34 protein are shown below each lane. Panel B, lysates of HEK293 cells expressing vector control (C), WT GADD34-FLAG (GADD34), or the mutant protein (Y262F) were subjected to immunoprecipitation (IP) using FLAG-M2 beads. * indicates a nonspecific band. Panel C, in separate experiments, lysates of HEK293T cells expressing the mutant GADD34-FLAG proteins, Y262F, Y262E, and Y262D, were incubated with either control agarose (C) or Microcystin-agarose (MC) as described in “Materials and Methods.” The bound proteins were eluted in sample buffer, separated by SDS-PAGE, and immunoblotted for GADD34 and PP1.