反应详情

HEK 293 cells were grown in 10 cm plates with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). On the day of the experiment, cells were incubated with low glucose medium supplemented with 0.5% FCS for 1 hour, followed by the addition of the conjugate L803-mts or its respective controls LE803-mts and LS803-mts at various concentrations, for additional 2.5 hours. A vehicle control of DMSO (0.1% DMSO) was also tested. Cells were thereafter washed twice with ice-cold GS buffer (50 mM Tris, pH=7.8, 100 mM NaF, 10 mM EDTA with protease inhibitors: 20 μg/ml leupeptine, 10 μg/ml aprotinine, 10 mg/ml pepstatin A, 1 mM benzamidine), scraped with the same buffer, and frozen in liquid nitrogen (as described in Eldar-Finkelman et al., 1996). Glycogen synthase activity was assayed according to the method of Thomas et al. (1968), based on the incorporation of uridine 5-diphosphate [14C] glucose (UDPG) into glycogen. Aliquots of cell lysates (15 μl) were incubated with 15 μl reaction mixture (66.6 mM Tris, pH=7.8, 32.5 mM KF, 0.8 μCi/μl [14C]-UDPG (400 μM), 13 mg/ml glycogen rabbit liver, Sigma) for 20 minutes at 30° C. (as described in Eldar-Finkelman et al., 1996). The reactions were then spotted on ET31 (Whatman) papers, washed with 66% ice-cold ethanol, and counted for radioactivity. Glycogen synthase assays were measured in the presence of 0.1 mM glucose-6-phosphate (G6P). Similar results were obtained when G6P was absent in the assays (data not shown).

1. concentrationand LS803-mts at various concentrations
2. waitfor additional 2.5 hours
3. washCells were thereafter washed twice with ice-cold GS buffer (50 mM Tris, pH=7.8, 100 mM NaF, 10 mM EDTA with protease inhibitors
4. temperaturefrozen in liquid nitrogen (as described in Eldar-Finkelman et al., 1996)
5. customAliquots of cell lysates (15 μl) were incubated with 15 μl reaction mixture (66.6 mM Tris, pH=7.8, 32.5 mM KF, 0.8 μCi/μl [14C]-UDPG (400 μM), 13 mg/ml glycogen rabbit liver, Sigma) for 20 minutes at 30° C. (as described in Eldar-Finkelman et al., 1996)
6. washwashed with 66% ice-cold ethanol
7. customSimilar results