FIGURE 3.

Substitution of tyrosine 262 with phenylalanine stabilizes human GADD34. Panel A, HEK293T cells expressing WT GADD34-FLAG (labeled WT-FLAG) or the mutant GADD34(Y262F)-FLAG (labeled Y262F-FLAG) were treated with 30 μg/ml cycloheximide (CHX). The cells were lysed at various time points in SDS sample buffer, and the samples subjected to SDS-PAGE and immunoblotting (IB) with anti-FLAG antibody. Immunoblotting with anti-tubulin antibody established equivalent protein loading in all lanes. Panel B, multiple independent immunoblotting experiments as described in panel A were quantified for either GADD34 or Y262F levels by scanning and the combined results are shown as a bar graph with standard errors. Panel C, HEK293T cells expressing WT GADD34-FLAG were treated with 30 μg/ml CHX in the presence and absence of 1 mm Na₃VO₄. Immunoblotting with anti-tubulin established equivalent protein loading. Panel D, quantitation of GADD34 protein in control and Na₃VO₄-treated cells in multiple independent experiments was undertaken and is shown as a bar graph with standard errors. Panel E, HEK293T cells expressing WT GADD34-FLAG or the mutants Y262F, Y118F, and S264A were exposed to 30 μg/ml CHX and at selected times, lysed in SDS sample buffer. Separated by SDS-PAGE and analyzed by immunoblotting.