p19^INK4dinteracts with and negatively regulates PEBP1 and PEBP1 links p19^INK4dwith the ERK pathway. (A) Representative images of silver-stained gels of IP proteins using IgG and p19^INK4d antibody. (Upper) Nonspecific bands; (lower) specific band is marked with a red arrow. (B) Representative electrospray ionization-MS and MS/MS profiling of a tryptic peptide that owns higher content. The top right corner is the amino acid sequence of this peptide and the specific amino acid sequence belongs to PEBP1 through identification of proteins from the protein database. (C) Representative immunofluorescence images showing p19^INK4d and PEBP1 localization in erythroblasts. DAPI was used to stain the nucleus. (D) Representative images of co-IP experiments with a p19^INK4d (left) or PEBP1 antibody (right). (E) Representative images of western blotting showing PEBP1 expression in erythroblasts infected with Lucif shRNA or p19^INK4d shRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. (F) Representative images of western blotting showing PEBP1, p-ERK, ERK, HSP70, and GATA1 levels in erythroblasts transfected with PEBP1 siRNA or scramble siRNA (left). Quantitative analysis of western blotting data from 3 independent experiments (right). GAPDH was used as a loading control and the results were normalized to GAPDH protein. Statistical analysis of data from 3 independent experiments and bar plot represents mean ± SD of triplicate samples. * P < .05, ** P < .01.