Biomedical Engineering Reference
In-Depth Information
Fig. 1
High-throughput screen identifi es Ube3C as a regulator of MET internaliza-
tion. (
a
) MET internalization was blocked in HGF-treated HeLa cells reverse
transfected with the siRNA Ube3C-3 and pooled Ube3C (1-3) siRNAs. The indi-
vidual Ube3C-1 or Ube3C-2 siRNAs or siRNAs (5, 11, and pooled) directed against
the non-ATPase regulatory subunit 4 (PSMD4) of the 26S proteasome had no
effect on MET internalization. Control represents internalized MET in cells treated
with nontargeting (NT) siRNA, which was set to 100 %. Pretreatment with
Dynasore inhibited MET internalization. Values represent mean internalized MET
levels ± SEM from triplicate experiments. (
b
) Western analysis confi rmed knock-
down of Ube3C in cells transfected with Ube3C-3 siRNA and not in untreated
cells or cells treated with a nontargeting (NT) siRNA
2. For internalization studies, the transfection mixture of
Ube3C-3 siRNA, NT siRNA, DharmaFECT 2, and 4×10
5
cells in 4 ml of DMEM containing 10 % FBS was left to adhere
onto 60-mm plates. After 24 h incubation, the cells were serum
starved for 24 h by incubation at 37 °C in DMEM only.
3. The following morning, the cells were incubated in prewarmed
DMEM supplemented with 100 ng/ml HGF at 37 °C for 15,
30, or 60 min, respectively, to promote MET internalization.
Controls included serum-starved cells that were treated with
media lacking ligand.
