Net1166005mc

Protein synthesis was measured by incorporation of Leucine L-(4,5-³H) (obtained from Perkin Elmer (NET1166005MC, Waltham, MA, USA). Cells were treated for drugs as indicated for 5 h and washed with PBS and RPMI-1640 media and 3.5 μCi/mL Leucine L-(4,5-³H) was added for 1 h. Cell suspensions were placed on Whatman GF/C microfiber filters and proteins were precipitated with ice-cold 5% trichloroacetic acid (TCA) (Sigma-Aldrich, St Louis, MO, USA). Filters were washed twice with 5% TCA, once with 99% ethanol, and left to dry. Radioactivity was measured using liquid scintillation counting.

Protein synthesis was measured by incorporation of Leucine L- (4,5-³H ) (NET1166005MC ) obtained from Perkin Elmer (USA, Waltham, MA). Prior to the treatment ALL cells were cultured in RPMI-1640 growth media with 10% FBS and 1% penicillin/streptavidin. Cells were treated for 5h with DMSO, 160 nM, 320 nM, 640 nM, 1280 nM of VLX1570, or for 3h with 160 nM VLX1570, 0,001 U L-asparaginase and combination of the two drugs. Subsequently cells were washed with PBS and RPMI-1640 media without L-leucine and 1μCi/mL Leucine L-(4,5-³H) was added for 1h. Cell suspensions were placed on Whatman GF/C microfiber filters and proteins were precipitated with ice-cold 5% trichloroacetic acid (TCA) (Sigma Aldrich). Filters were washed twice with 5% TCA, once with 99,5 % ethanol and left to dry. Radioactivity was measured with liquid scintillation counter. Incorporation increased linearly over time (15 – 60 minutes).