Rheb activation kit

The RheB activation assay was performed using a RheB activation kit (ab173243, Abcam) according to the manufacturer's protocol. Briefly, Vector, WT_118 and H868R_07 cells were cultured in 10 cm dishes until they reached 90% confluence (~10⁷ cells). Following washing with ice-cold PBS, the cells were lysed in 0.5 ml 1X ice-cold Assay/Lysis Buffer. Whole cell lysates (WCL) obtained from Vector, WT_118, and H868R_07 were subjected to the assay. WCL were incubated with 1 μL anti-active RheB mouse monoclonal antibody and 20 μL protein A/G agarose bead slurry at 4°C for 1 hour with gentle agitation. Beads were pelleted by centrifugation, washed 3 times with Lysis Buffer and then subjected to SDS-PAGE analysis. RheB was detected by Western blot analysis using a rabbit antibody directed against RheB-GTP. The samples of positive and negative controls were generated according to manufacturer's protocol.

NIH3T3 cells and COS-7 cells were purchased from American Type Culture Collection (ATCC) and used within six months after cells were ordered. These cell lines were not authenticated in our laboratory. Rapamycin, 5-Bromo-2′-deoxyuridine (BrdU) and cycloheximide were obtained from Sigma. PF-4708671 was purchased from Selleckchem. MG132 was purchased from CalBiochem. Antibodies directed against Myc-tag (9E10), Flag-tag, and HA-tag were obtained from Covance. Antibodies directed against VPS34 (cat#4263), VPS15 (cat#14580), Beclin 1 (cat#3495), pT389-S6K1 (cat#9234), S6K1 (cat#2708), pT37/46-4EBP1 (cat#2855), 4EBP1 (cat#9644), pS235/236-S6 (cat#4858), S6 (cat#2217), pErk1/2 (cat#4370), Erk1/2 (cat#4695), pS473-Akt(cat#4060), Akt (cat#4685), RheB (cat#13879), TSC1 (cat#6935), TSC2 (cat#4308), BrdU (cat#5292), and Cyclin E (cat#4129) were purchased from Cell Signaling Technology. Fluorescently tagged antibodies were obtained from Invitrogen. The class III PI3K lipid kinase ELISA kit was purchased from Echelon Biosciences. The RheB activation kit was purchased from Abcam. 4-6 weeks old SCID mice were ordered from National Cancer Institute (NCI), NIH (Bethesda, MD).