Figure 1. Ptsn associates with the MIT... | Rockefeller University Press

Figure 1.

$Ptsn associates with the MIT complex. (A–D) Schematic of a split tandem affinity purification method to isolate the proteins associated with the MIT complex (A). 1: U2-OS cells stably expressing integrin αIIb-SBP and β3(D119A) to form ligand binding–defective αIIb(SBP)β3(D119A) were transfected with a plasmid encoding Flag-tagged RIAM (B), Flag-tagged LPD (C), or no insert (D). 2: Cell lysis. 3: Anti-Flag affinity chromatography of cell lysates. 4: Native proteins were eluted with 200 μg/ml Flag peptide. 5: The eluate was passed through a streptavidin column. Bound MIT complexes were washed, eluted with 5 mM biotin, and separated by SDS-PAGE. Both RIAM– and LPD–MIT complexes contain Ptsn. All fractions including whole-cell lysate (0.5% of total), anti-Flag flow-through (FT, 0.5%), Flag eluate (10%), streptavidin FT (10%), and streptavidin eluate (50%) were analyzed by immunoblotting. Results are representative of three independent experiments. Source data are available for this figure: SourceData F1.$

Ptsn associates with the MIT complex. (A–D) Schematic of a split tandem affinity purification method to isolate the proteins associated with the MIT complex (A). 1: U2-OS cells stably expressing integrin αIIb-SBP and β₃(D119A) to form ligand binding–defective α_IIb(SBP)β₃(D119A) were transfected with a plasmid encoding Flag-tagged RIAM (B), Flag-tagged LPD (C), or no insert (D). 2: Cell lysis. 3: Anti-Flag affinity chromatography of cell lysates. 4: Native proteins were eluted with 200 μg/ml Flag peptide. 5: The eluate was passed through a streptavidin column. Bound MIT complexes were washed, eluted with 5 mM biotin, and separated by SDS-PAGE. Both RIAM– and LPD–MIT complexes contain Ptsn. All fractions including whole-cell lysate (0.5% of total), anti-Flag flow-through (FT, 0.5%), Flag eluate (10%), streptavidin FT (10%), and streptavidin eluate (50%) were analyzed by immunoblotting. Results are representative of three independent experiments. Source data are available for this figure: SourceData F1.