Tyr phosphorylation of RBC membrane protein is increased in ChAc compared with normal controls. RBCs from control (C) and ChAc subjects were fractionated as described in “Study design” and in the legend of Figure 1B-E. Western blot (Wb) analysis with specific anti–phosphotyrosine (PY) antibodies of RBC membrane proteins separated by either bidimensional electrophoresis (2DE) or monodimensional electrophoresis (1DE). (A) Twin 2DE gels were run: one used for colloidal Coomassie-stained gels (Figure 1D) and the other for the Western blot analysis with specific anti–phosphotyrosine (PY) antibodies. Top panel: Membranes of high-density RBC fraction (F2) from control (C) and ChAc subjects. Similar results were also obtained in low-density RBC fraction (F1) from control and ChAc (data not shown). Bottom panel: Dephosphorylation of blotted proteins by recombinant λ protein phosphatase (400 U/mL). The blotted membranes were incubated in TBS containing 1% BSA, 0.1% Triton X-100, 2mM MnCl₂ (overnight at 4°C), and then probed with anti–phosphotyrosine antibodies. The data are representative for 3 experiments with similar results. (B) 1DE gels (13 cm) were blotted for Western blot (Wb) analysis with specific anti–phosphotyrosine (PY) antibodies. The bands with different staining intensities were identified by mass spectrometry (Table 2; “Comparative proteomic analysis”; supplemental Methods). Actin was used as a loading control. The data are representative of 8 experiments. See also supplemental Figure 1B for densitometric analysis of the Tyr phosphorylation profile of the RBC membrane proteins.