American Society of Hematology

Figure 4

$Figure 4. SWAP-70 phosphorylation regulates its binding to F-actin. (A) Tyrosine 517 is important for F-actin binding. Either the Wt C-terminus or Y517A mutant C-terminus were incubated with decreasing amounts of F-actin, then centrifuged such that the F-actin sediments and F-actin binding proteins bound cosediment. The remaining soluble proteins were also collected. The figure shows the Coomassie-stained SDS-PAGE gel of the sedimented and soluble proteins. The bar chart represents average values (± SD of 5 independent experiments) of the percentage of SWAP-70 found in the insoluble fraction as determined by densitometery. (B) Phosphorylation of SWAP-70C reduces its binding to F-actin. To ascertain the effect of SWAP-70 phosphorylation on F-actin binding, the F-actin binding assay was repeated with the use of phosphorylated or nonmodified SWAP-70C. The bar graph shows the percentage of phosphorylated and nonphosphorylated SWAP-70C found in the insoluble fraction as determined by densitometery (n = 4). (C) SWAP-70 Y517F is enriched in an insoluble cytoskeletal fraction in vivo in comparison to Wt SWAP-70. The potential effect of phosphorylation on F-actin binding in vivo was measured by infecting Swap70−/− cells with retroviruses expressing either wt or a SWAP-70 Y517F mutant. After 72 hours, cell extracts were made and the insoluble fraction containing the cytoskeleton was collected by centrifugation. Proteins present in the soluble, insoluble, and total extracts were determined by Western blotting with the antibodies indicated. The ratio of each form of SWAP-70 relative to actin in the insoluble fraction was used to calculate the percentage of insoluble SWAP-70 compared with the same ratio in the unfractionated total preparation and plotted as shown (n = 5).$

SWAP-70 phosphorylation regulates its binding to F-actin. (A) Tyrosine 517 is important for F-actin binding. Either the Wt C-terminus or Y517A mutant C-terminus were incubated with decreasing amounts of F-actin, then centrifuged such that the F-actin sediments and F-actin binding proteins bound cosediment. The remaining soluble proteins were also collected. The figure shows the Coomassie-stained SDS-PAGE gel of the sedimented and soluble proteins. The bar chart represents average values (± SD of 5 independent experiments) of the percentage of SWAP-70 found in the insoluble fraction as determined by densitometery. (B) Phosphorylation of SWAP-70C reduces its binding to F-actin. To ascertain the effect of SWAP-70 phosphorylation on F-actin binding, the F-actin binding assay was repeated with the use of phosphorylated or nonmodified SWAP-70C. The bar graph shows the percentage of phosphorylated and nonphosphorylated SWAP-70C found in the insoluble fraction as determined by densitometery (n = 4). (C) SWAP-70 Y517F is enriched in an insoluble cytoskeletal fraction in vivo in comparison to Wt SWAP-70. The potential effect of phosphorylation on F-actin binding in vivo was measured by infecting Swap70^−/− cells with retroviruses expressing either wt or a SWAP-70 Y517F mutant. After 72 hours, cell extracts were made and the insoluble fraction containing the cytoskeleton was collected by centrifugation. Proteins present in the soluble, insoluble, and total extracts were determined by Western blotting with the antibodies indicated. The ratio of each form of SWAP-70 relative to actin in the insoluble fraction was used to calculate the percentage of insoluble SWAP-70 compared with the same ratio in the unfractionated total preparation and plotted as shown (n = 5).

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