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. Author manuscript; available in PMC: 2009 Mar 1.
Published in final edited form as: Mol Cancer Res. 2008 Mar;6(3):501–508. doi: 10.1158/1541-7786.MCR-07-2075

FIGURE 4.

FIGURE 4

Polysome analysis of cell lines expressing low (PC3 and DuPro) and high (LNCaP and C4-2) levels of MKK4 protein. A. Cytoplasmic cell lysates were separated in sucrose density gradients and fractions were collected. Fraction 1 corresponds to the lightest fraction; fraction 12, the heaviest. Total RNA was prepared from each fraction, separated by gel electrophoresis, and transferred to membranes for Northern blotting with a probe spanning the MKK4 coding region. Membranes were stripped and reprobed to monitor the expression of glyceraldehyde-3-phosphate dehydrogenase mRNA (encoding a housekeeping gene) in these fractions. Membranes were subsequently stripped and reprobed for expression of β-actin mRNA, encoding a second housekeeping gene. These two genes had strikingly similar mRNA patterns, supporting their use as a measure of actively translating polysome fractions. Representative data from three experiments are shown. B. Graphic representative of polysome analysis. The two pairs of low MKK4–expressing versus high MKK4–expressing cells were arbitrarily selected (PC3 versus LNCaP and DuPro versus C4-2). Pooled fractions are indicated as follows: fractions 1 and 2 (A), fractions 2 and 3 (B), fractions 4 and 5 (C), etc.