FIGURE 2.
GADD34 expression in HepG2 cells during thapsigargin and actinomycin D treatment. A, Northern blots of RNA and (C) immunoblots of lysates from HepG2 cells alone (unt) or treated with 5 μg/ml actinomycin D (actD), 1 μm thapsigargin (thap), or thapsigargin/actinomycin D (actD/thap) for the indicated times. A, radiolabeled probes specific to GADD34, ATF4, CHOP, and GAPDH mRNAs (indicated to the right) were quantitated (B) using phosphorimaging analysis and normalized to the amount of RNA at the 3-h untreated time point. C, GADD34, ATF4, phosphorylated eIF2α, total eIF2, and CHOP were detected by antibodies as indicated to the right. Representative Western blots of at least three independent experiments are shown. For GADD34, two independent experiments are shown, which shows reproducible induction of GADD34 protein levels under thapsigargin alone and thapsigargin and actinomycin D treatment. The arrow indicates the migration of GADD34 protein, and the asterisk indicates nonspecific proteins recognized by the GADD34 antibody. D, quantitation of a representative GADD34 immunoblot. The amount of GADD34 protein was normalized to the amount of GADD34 at the 3-h untreated time point (Odyssey-Licor). E, polysomal Northern blot analysis of RNA in fractions from untreated HepG2 cells (unt) or from cells after a 30-min treatment with 1 μm thapsigargin (thap), 5 μg/ml actinomycin D (actD), or 1 μm thapsigargin (actD/thap) as indicated to the right. The distribution of GADD34 and GAPDH mRNAs is shown by Northern blot analysis using radiolabeled DNA probes. Fractions from top to bottom of the gradient are represented from left to right, respectively. Locations of 80 S and polysomes across the gradient are indicated at the bottom. Quantitation of the amount of radioactivity within each fraction is shown in supplemental Fig. S1.