Proteomic and Phospho-Proteomic Profile of Human Platelets in Basal, Resting State: Insights into Integrin Signaling (original) (raw)
Figure 3
Platelets stored without citrate buffer show Talin degradation upon thawing.
A. Coomassie stained 4–12% Bis-Tris SDS-PAGE gel shows selective degradation of Talin in lysates from platelet pellets stored in the absence of citrate buffer (Lane 3) Vs no degradation in the presence of citrate (Lane 2). B. Immuno-blotting with anti-Talin antibody against the Talin N-terminal rod region (antibody 8D4, [27]) shows that Talin is degraded to smaller fragments in three independent platelet samples stored in the absence of citrate buffer (Lanes 4–6) as compared to three independent platelet samples stored in the presence of citrate (lanes 1–3). Notice the slight reduction in the MW of full-length Talin band in lanes 4–6 as well as the presence of ∼37 kDa fragments in lanes 4–6. C. Similarly, immuno-blotting with anti-Talin antibody against the Talin C-terminal region (antibody C20) also shows that Talin is degraded to a smaller MW species in two independent platelet samples stored in the absence of citrate buffer (Lanes 3–4) as compared to two independent platelet samples stored in the presence of citrate (lanes 1–2). Protein MW markers are as labeled. D. Immuno-blotting with the anti-integrin β3 mAb shows no degradation of integrin β3 upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled. E. Immuno-blotting with the anti-β-actin mAb shows no β-actin degradation upon thawing of stored platelet pellets. Platelet lysates from three independent platelet samples stored either in the presence of citrate (lanes 1–3) or absence of citrate (lanes 4–6) were thawed and analyzed by 4–12% Bis-Tris 1D SDS PAGE followed by western blotting. Protein MW markers are as labeled.