Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes
Figure 5
Hem1−/− erythrocytes contain increased phospho-adducin and decreased levels of essential membrane skeletal proteins.
SDS-PAGE analysis of purified erythrocyte ghosts was used to evaluate relative levels of erythrocyte membrane skeletal proteins between WT and Hem1−/− mice. (A) Coomassie-stained polyacrylamide gel (representative of 6 animals per genotype). Lanes contain equivalent amounts of total protein. β-actin immunoblot is shown below. (B) Immunoblots of erythrocyte membrane ghosts from purified WT and Hem1−/− erythrocytes, loaded according to total protein and equivalent β-actin. Each pair is representative of results for at least 5 individuals of each genotype. (C) Activation of PKC results in increased phosphorylation of adducin on Serine 724. Purified WT and Hem1−/− erythrocytes were stimulated with the phorbol ester PMA (20 ng/ml) and were harvested and lysed at the indicated timepoints post-stimulation. A representative immunoblot of three separate experiments is shown (3 animals per genotype). (D) Erythrocyte ghosts from Hem1−/− mice contain increased PP2A catalytic subunit (PP2Ac) and decreased PP2A regulatory subunit (PP2Ar) protein relative to WT erythrocytes. Shown are total protein levels determined by immunoblot. Numbers below each scan represent relative protein expression levels in Hem1−/− versus WT samples. Samples were loaded according to levels of total protein. (E) WT and Hem1−/− erythroblasts were stimulated in Okadaic acid (OA) and were harvested and lysed at the indicated timepoints post-stimulation. A representative immunoblot of 3 separate experiments is shown. Samples were loaded based on equivalent cell number.