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. 2008 Sep 15;28(23):6989–7000. doi: 10.1128/MCB.00724-08

FIG. 5.

FIG. 5.

GADD34 is polyubiquitinated. (A) HeLa cells expressing either FLAG(109)GADD34 (lanes 1 and 2), FLAG-RFP (lane 3), GADD34-GFP (lane 4), or both FLAG-RFP and GFP-GADD34 (lane 5) were treated with or without MG262 (5 μM, 8 h). Cell lysates were subjected to immunoprecipitation using agarose-conjugated anti-FLAG antibody. Lysates and immunoprecipitates were subjected to SDS-PAGE and immunoblotted with antiubiquitin, anti-FLAG, and anti-GFP antibodies. Asterisks indicate the location of heavy and light chains of anti-FLAG IgG in all immunoprecipitates. Molecular mass markers (in kilodaltons) migrated as indicated on each blot. (B) GADD34-GFP was expressed in HEK293T cells in combination with empty vector, with vector encoding wild-type myc-tagged ubiquitin (myc-Ub), or with a mutant myc-ubiquitin (myc-Ub K48R). After 24 h, protein synthesis was inhibited with CHX, and GADD34 levels analyzed at the indicated times by immunoblotting. Tubulin levels established equivalent protein loading. (C) The results from three independent experiments (shown in panel B) were quantified by laser scanning and are shown with standard errors.