Metabolic Labeling of Erythroid Cells

Metabolic labeling of proteins was carried out using Click-iT chemistry^{12 (link)}. CD34⁺ HSPCs derived from adults and newborns were cultured in parallel until day 7 of erythroid differentiation washed with warm PBS, and incubated in methionine-free RPMI medium (Sigma Aldrich) with 10% FBS and 2 mM L-glutamine for 1 hour at 37 °C to deplete both cell types of methionine reserves. Methionine-starved cells were then labeled with Click-iT L-azidohomoalanine (L-AHA, Life Technologies) at a concentration of 50 μM for 6 hours at 37 °C. Harvested cells were washed twice in PBS warmed to 37 °C, and protein extraction was carried out in RIPA lysis buffer supplemented with PMSF, sodium orthovanadate, and protease inhibitor cocktail solution (Santa Cruz Biotechnology). 50 μg of protein lysate was used for the Click reaction with tetramethylrhodamine alkyne (TAMRA, Life Technologies) in a total volume of 200 μl using the Click-iT Protein Reaction Buffer Kit (Thermo Fisher Scientific). TAMRA signal was detected on total proteins separated by SDS-gel electrophoresis with an AI600 imager (Amersham Biosciences).