Spectrin isoforms in megakaryocytes and platelets. (A) Immunoblot showing the presence of erythroid and nonerythroid spectrin isoforms in MKs and platelet lysates. Isoform-specific antibodies were used to identify α1, β1, α2, and β2 spectrins in MKs at different stages of maturation and in platelets (Plt). Accordingly, α2 and β2 antibodies failed to recognize the erythroid spectrin isoforms in lysates of erythrocyte ghosts, whereas α1 and β1 antibodies identified the erythroid spectrin isoforms in the erythrocyte ghosts. Murine fibroblasts (3T3 Swiss) were used as a negative control for α1 and β1 spectrins. GAPDH was used as a loading control. Blots show anti-spectrin isoform labeling during different days of megakaryocyte culture: FLCs (day 0) and MKs at different stages: day 2, young MKs; day 3, MKs just before producing proplatelets; day 4, MKs after producing proplatelets. (B) Immunoblot analysis showing the distribution of spectrin isoforms in the pelleted (P) actin cytoskeleton and soluble (S) fractions of FLCs, MKs, and platelets. A higher fraction of αII and βII spectrin isoforms associated with the cytoskeletons in MKs just before making proplatelets (day 3), compared with other MK stages. Western blots from 3 different experiments were quantified by densitometry (supplemental Figure 6). (C) Quantitative PCR. The relative mRNA expression (compared with GAPDH) of spectrin isoforms in MKs determined by quantitative RT-PCR. Nonerythroid spectrins (α2 and β2 spectrins) were expressed at higher levels than erythroid isoforms (α1 and β1 spectrins) in MKs.