Low levels of MKK4 protein do not correlate with genomic deletions or low levels of MKK4 mRNA. A. MKK4 protein expression in a panel of prostate (CaP), ovarian, and cervical cancer cell lines. Protein (20 or 100 μg) was immunoblotted for MKK4. Rat brain (5 μg) and ASPC-1 (20 or 100 μg) cell protein served as positive and negative controls for MKK4 expression, respectively. β-Actin was used as a loading control. Immunoblots are representative of data from six independent experiments. B. Genomic DNA was isolated and PCRs were done with nested primers specific for each of the 11 exons (exons A-E) of the MKK4 gene. Human genomic DNA was used as a positive control for all reactions. Water and ASPC-1, a pancreatic cancer cell line harboring a homozygous deletion of MKK4 exons B and C, served as negative controls. Data are representative of two independent experiments. C. MKK4 mRNA expression in cancer cell lines. Polyadenylated mRNA was isolated from total RNA for Northern blotting with a probe complementary to the MKK4 coding region. Rat brain and ASPC-1 cells served as positive and negative controls for MKK4 expression, respectively. Representative data from three independent experiments are shown.