FIGURE 6.

HuR-D226A mutant acts in a dominant-negative manner during stress. A, measurement of HuR in normoxic (no CoCl₂) or hypoxic cells (with CoCl₂) is shown. UM74B cells were transfected with indicated plasmid vectors (GFP, GFP-HuR, and GFP-D226A) followed by 36 h of hypoxic treatment, and cell extracts (50 μg) were analyzed by Western blotting using antibodies against HuR and the β-actin antibody. All cells were normalized based on GFP counts using flow cytometry. NS represents a nonspecific band. B, shown is the growth rate of representative plasmids transfected after 48 h. Cells were plated at a density of 10³ cells per ml and counted at 48 h. All the transfection vectors were normalized by GFP cell sorting using flow cytometry. Triplicate samples were counted at each time point, and the S.D. values were used to plot the error bars (*, p < 0.05, n = 3).