RNA and protein analysis of the β-adducin −/− mice.

(A) Northern blot analysis of brain and spleen β-adducin mRNA from adult wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice. The arrows indicate the different β-adducin mRNA species detected. (B) Methylene blue staining of the membrane utilized in A. (C) The signals obtained in A were normalized against the 18S RNA content present in the membrane. RNA levels are expressed as a percentage of the RNAs of wild-type mice for each tissue. (D) Western blot analysis of brain (lanes 1-6) and spleen (lanes 7-9) protein extracts (30 μg) from adult wild-type (+/+), heterozygous (+/−), and homozygous mutant (−/−) mice using a rabbit anti-mouse β-adducin Δ9-13 polyclonal antibody. Lanes 4-6 are a 15× overexposure of the blot shown in lanes 1-3. The β-adducin (β-Add97 form) bands are marked by black arrows. (E) Western blot analysis of ghost proteins (lanes 1-3, 0.7 μg; lane 4, 10 μg), bacteria purified recombinant mouse β-adducin Δ9-13 (lanes 8 and 9, containing 2 and 5 ng of purified protein, respectively), and RBC cytoplasmic proteins (lanes 10-12, 100 μg) was revealed with the same antibody utilized in panel D. Lanes 1 to 3 are a lower exposure of the blot shown in lanes 5 to 7. The β-adducin (β-Add97 form) and the recombinant β-adducin Δ9-13 product (Rec.) are marked by a black arrow and a black dot, respectively. The lower molecular weight bands seen in lane 10 are marked by gray arrows.