iTRAQ™ validation by immunoblot. Equal quantities of ghost lysates were pooled from 6 to 8 donors per group (control vs Mi.III) for IP, and one-tenth of the pulldown (vol/vol) was loaded onto 4% to 12% SDS-polyacrylamide gel electrophoresis for immunoblot comparison. (A) Immunoblots confirmed that glucose transporter type I, ANK1, EPB42, α spectrin, and β spectrin were not quantitatively different in AE1 immunoprecipitates from both groups. The pulldown experiments were repeated 2 to 8 times, and IP was also confirmed using another anti-AE1, BRIC170 (data not shown). (B) Both anti-4.1R and anti-Rh antibodies pulled down GPA, GPB, Gp.Mur, and AE1, indicating that 4.1R and Rh polypeptides were part of the AE1-based complexes. (C) Rh-associated glycoprotein was coimmunoprecipitated with AE1. AE1 IP was repeated and confirmed with both anti-AE1 (AE12-M and BRIC170). (D) AQP1 was more substantially associated with AE1 on Mi.III⁺ membrane. (Left) AQP1 immunoblot of the pooled ghost lysates showed similar expression levels for both Mi.III and the control. (Right) Both anti-AE1 antibodies coimmunoprecipitated more AQP1 from Mi.III. The coimmunoprecipitation experiment has been repeated and confirmed 7 times.