Figure 5
Figure 5. Mechanism of weakened erythrocyte junctional complex. (A) Fairbanks gel electrophoresis (4% acrylamide gel, denaturing conditions) of ghosts. Band 3 was used to normalize protein loading. (B) Spectrin dimer-tetramer equilibrium analysis. Spectrin tetramers were extracted at low ionic strength buffer at 0°C (on ice; upper gel panel) and converted to dimers at 37°C (lower gel panel). Dimer-tetramer content was analyzed by Fairbanks gel electrophoresis under nondenaturing conditions. Note that the spectrin tetramers were dissociated (>95%) into dimers in dematin FLKO (−/−) sample, indicating that dematin loss does to affect spectrin self-association under these conditions. “T” indicates position of spectrin tetramers. “D” indicates position of spectrin dimers. (C) Coimmunoprecipitation of dematin using Flag-β-adducin. HEK293T cells were cotransfected with constructs encoding dematin and Flag-β-adducin (Add2) or empty vector. Immunoprecipitation was performed with anti-Flag agarose beads. Dematin amount in the immune-complex was normalized by western blotting using the anti-dematin polyclonal antibody. (D,F,G) GST pull-down assays. T, Trx tag alone; F, Trx-dematin-rD full length; S, Trx-dematin-rD-S381E mutant; H, Trx-Dematin-headpiece domain; C, Trx-Dematin-core domain-rD; G, GST tag alone; A, GST-β-Adducin; β, GST-β-Spectrin-actin-binding domain1-2; α, GST-α-Spectrin-20-21-EF. Equal loading of GST or GST fusion proteins was confirmed by Coomassie staining. Black dots represent phosphorylation-mimic mutation where serine-381 was changed to glutamic acid (E) in the dematin headpiece domain. Dematin-S381E change promotes dematin headpiece interaction with the core domain. (E) Dematin far-western blotting. Human erythrocyte ghosts were transferred onto nitrocellulose membrane, and blots were incubated with purified human dematin alone or in the presence of purified spectrin. No signal was detected with preimmune serum (lane 1). Anti-dematin polyclonal antibody detected endogenous 48- and 52-kDa polypeptides of dematin (lanes 2 and 3), as well as purified dematin bound to β-spectrin (lane 2). Note that dematin binding to immobilized β-spectrin was eliminated by soluble spectrin (lane 3). Dematin did not bind to α-spectrin under these conditions.

Mechanism of weakened erythrocyte junctional complex. (A) Fairbanks gel electrophoresis (4% acrylamide gel, denaturing conditions) of ghosts. Band 3 was used to normalize protein loading. (B) Spectrin dimer-tetramer equilibrium analysis. Spectrin tetramers were extracted at low ionic strength buffer at 0°C (on ice; upper gel panel) and converted to dimers at 37°C (lower gel panel). Dimer-tetramer content was analyzed by Fairbanks gel electrophoresis under nondenaturing conditions. Note that the spectrin tetramers were dissociated (>95%) into dimers in dematin FLKO (−/−) sample, indicating that dematin loss does to affect spectrin self-association under these conditions. “T” indicates position of spectrin tetramers. “D” indicates position of spectrin dimers. (C) Coimmunoprecipitation of dematin using Flag-β-adducin. HEK293T cells were cotransfected with constructs encoding dematin and Flag-β-adducin (Add2) or empty vector. Immunoprecipitation was performed with anti-Flag agarose beads. Dematin amount in the immune-complex was normalized by western blotting using the anti-dematin polyclonal antibody. (D,F,G) GST pull-down assays. T, Trx tag alone; F, Trx-dematin-rD full length; S, Trx-dematin-rD-S381E mutant; H, Trx-Dematin-headpiece domain; C, Trx-Dematin-core domain-rD; G, GST tag alone; A, GST-β-Adducin; β, GST-β-Spectrin-actin-binding domain1-2; α, GST-α-Spectrin-20-21-EF. Equal loading of GST or GST fusion proteins was confirmed by Coomassie staining. Black dots represent phosphorylation-mimic mutation where serine-381 was changed to glutamic acid (E) in the dematin headpiece domain. Dematin-S381E change promotes dematin headpiece interaction with the core domain. (E) Dematin far-western blotting. Human erythrocyte ghosts were transferred onto nitrocellulose membrane, and blots were incubated with purified human dematin alone or in the presence of purified spectrin. No signal was detected with preimmune serum (lane 1). Anti-dematin polyclonal antibody detected endogenous 48- and 52-kDa polypeptides of dematin (lanes 2 and 3), as well as purified dematin bound to β-spectrin (lane 2). Note that dematin binding to immobilized β-spectrin was eliminated by soluble spectrin (lane 3). Dematin did not bind to α-spectrin under these conditions.

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