Function of (Ser169 → Ala) mutant p45 in the p45-deficient MEL cell variant CB3. P45-deficient CB3 cells were stably transfected with expression vectors encoding either wild-type (W) or (Ser169 → Ala) mutant (M) p45 and single G418-resistant colonies were selected as described in Materials and Methods. Cells were cultured for 72 hours in 4 mmol/L HMBA. (A) EMSAs were performed with 10 μg of nuclear extract protein and 10 fmol of NF-E2 oligodNT as described in Fig 2. The amount of NF-E2/oligodNT complexes formed correlated closely with the amount of p45 detected on Western blots (not shown). (B) Northern blots prepared with 8 μg of total cytoplasmic RNA were probed with a β-globin cDNA probe as described in Materials and Methods. Equal loading of the RNA was confirmed by ethidium fluorescence of the ribosomal RNA bands (not shown). Several representative clones expressing variable amounts of wild-type (W1-W6) or (Ser169 → Ala) mutant p45 (M1-M5) are shown; control MEL cells expressing endogenous p45 (C) are shown in the first lane for comparison.
