FIGURE 5.

C/EBPβ mRNA from βwt and βd/s cell lines. A, total RNA was isolated with respect to time after removal of doxycycline from the culture medium and exposure of the cells to the differentiation protocol. Accumulation of total C/EBPβ mRNA was determined by real-time PCR analysis as described under “Experimental Procedures.” The experiment was performed three times with identical results. B, cytosolic accumulation of C/EBPβ mRNA in βwt (Bw/t) and βd/s (Bd/s) cell lines is shown. The cytosolic fraction (cyto) of the cells was isolated using the Pierce NE-PER™ kit as we have described previously (29). Cytosolic RNA was isolated using the TRIZol™ reagent. Accumulation of C/EBPβ mRNA was determined by real-time PCR analysis as described under “Experimental Procedures.” The experiment was performed three times with similar results. Data are plotted as the mean ± S.D. C, the levels of βwt and βd/s mRNAs in the cytosol at the 48-h time point (five independent determinations) are shown. Normalization was to β-laminin mRNA, the expression of which does not change over the time course of the experiment, followed by correction for endogenous expression as discussed under “Experimental Procedures.” βwt content was defined as 100%, and βd/s is expressed relative to that value.