Identification of RBC band 3 binding to PfGARP using pull-down assay. TRX–PfGARP-M2 was used to pull down potential binding proteins from detergent-solubilized RBC ghosts. (A) Coomassie blue–stained 12% gel (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) showing TRX and TRX–PfGARP-M2 attached to cobalt beads. (B-C) TRX and TRX–PfGARP-M2 attached to beads were incubated with C₁₂E₈ solubilized normal untreated (NU) and chymotrypsin-treated (NC) ghosts. Bound proteins were transferred to nitrocellulose membrane. (Ponceau S staining). (C) Anti-band 3 western blotting. The 70-kDa band specifically associated with TRX–PfGARP-M2 in untreated RBCs (left panel). The 60-kDa and 70-kDa bands were detected in the chymotrypsin-treated RBCs (right panel). No binding of band 3 was detected with TRX-bound beads. (D) Western blot of C₁₂E₈-solubilized normal and chymotrypsin-treated ghosts as inputs. Western blotting of TRX–PfGARP-M2 and TRX proteins alone did not show any nonspecific signal detected by anti-band 3 antibody (data not shown).