ChAc RBCs show increased Lyn Tyr kinase associated with the membrane. RBCs from control (C) and ChAc were fractionated as described in “Study design” and in the legend of Figure 1B-E. (A) Western blot analysis with specific antibodies of membrane-associated Tyr kinase Lyn and phospho-Lyn (p-Lyn), Syk, and phospho-Syk (p-Syk). Actin was used as a loading control. Shown is a representative of 6 experiments. Vertical line(s) have been inserted to indicate a repositioned gel lane. (B) Western blot (Wb) analysis with specific antibodies against Tyr-8 on the N-terminal of band 3, as a Syk target, and Tyr-904 on the transmembrane domain of band 3 (as a Lyn target). We used normal RBCs treated with Na-vanadate (see “Study design”) as positive controls. Total band 3 was used as loading control. (C) Western blot (Wb) analysis with specific anti–phosphotyrosine (PY) antibodies of high-density fraction (F2) of RBCs from control (C) and ChAc subjects are shown. The F2 RBCs were incubated with or without the Src family kinase inhibitors PP1 (10μM) and PP2 (10μM) as previously reported.¹⁹  The arrows indicated the bands affected by PP1-PP2 treatment in ChAc RBCs compared with untreated ChAc RBCs. The data are representative of 3 experiments (on 6-cm gels). Actin was used as a loading control. The data are representative of 3 experiments. Similar results were also obtained with cells from low density fraction (F1) control and ChAc RBCs (data not shown).