Phosphorylation of protein 4.1 is induced via the ERK signaling pathway. Sham-treated or U0126-treated SS and normal (AA) RBCs ghosts coincubated with or without recombinant active ERK2 (ERK2) were enriched in phosphopeptides, followed by a label-free quantitative phosphoproteomics analysis. Treatment of SS RBCs with U0126 caused a significant decrease in doubly phosphorylated peptide within protein 4.1. The addition of ERK2 to the U0126-treated SS RBC ghosts increased the abundance of this phosphopeptide back to levels observed in untreated SS RBCs. The complementary trend for this phosphorylated peptide was also observed on the addition of ERK2 to AA RBCs sham-treated or U0126-treated.