Quantifying Protein Synthesis via HPG Labeling

To analyze protein synthesis activity, cellular incorporation of L-homopropargylglycine (HPG), a methionine analogue with alkyne moiety was detected by Click-iT reaction with azide-modified Alexa 488^{40 (link)}. Cells treated with HHT, imatinib or sunitinib for 1 h or 8 h were labelled with 25 µM HPG (Invitrogen, C10102) for the last 30 min of the drug treatment in L-methionine-depleted medium containing 5% dialyzed fetal calf serum (FCS; Gibco). Harvested cells were washed with PBS, fixed in 10% buffered formalin (4% formaldehyde) for 5 min at room temperature and permeabilized for 30 min in PBS containing 0.1% saponine (Sigma) and 1% FBS (HyClone). The Click-iT reaction was performed for 30 min at room temperature by suspending the cells in Click-iT cell reaction buffer containing 1 mM azide-modified Alexa 488 and 2 mM CuSO4 (Invitrogen). Cells were washed twice in PBS with 1% FCS, and the cell suspension was subjected to flowcytometry analysis. The protein synthesis inhibitor cycloheximide (100 µg/ml) was used as a positive control in this assay. Cells without Click-iT reaction were used as a background control.