0.45 m filter

The shRNA sequences were designed and cloned into the lentivirus vector pLKO.1-puro according to The RNAi Consortium's recommendation (http://www.broadinstitute.org/rnai/trc). The shRNA sequences are in the Supplemental Material. Lentiviruses were produced by cotransfecting individual shRNA constructs with the packaging plasmids pMDLg/pRRE and pRSV-Rev and the envelope plasmid pMD2.G into 293T cells using FuGENE 6 transfection reagent (Roche). Supernatants containing lentivirus particles were collected and filtered through a 0.45-µm filter (Thermo). T-ALL cells were infected with lentivirus in the presence of 8 µg/mL polybrene (Millipore) by centrifugation at 1300 rcf for 1.5 h. The infected cells were selected with 0.7 µg/mL puromycin (Sigma) in RPMI-1640 medium for at least 36 h after infection.

To silence βTrCP1 or ERK2 in HEK293T, lentiviral expression plasmids of pLenti-sh-ERK2 (Dharmacon, Lafayette, CO, USA) were co-transfected into HEK293T cells with psPAX2 and pMD2.G (Addgene, Cambridge, MA, USA) as indicated by the manufacturer’s recommended protocols. At 24 hours and 48 hours after transfection, we obtained a Lenti-sh-ERK2 medium containing viral particles from the HEK293T cells. The medium was filtered with a 0.45 µm filter (Cat. #: 723-2545, Thermo Fisher Scientific, Waltham, MA, USA) and used, with 1 to 2 µg/mL of polybrene, to infect HEK293T. After a maximum of 16 hours, the cell medium was exchanged with fresh complete medium. After 48 hours of maintenance, non-infected control cells were killed over a period of 3 days by treatment with 2 μg/mL of puromycin (Cat. #: A111308, Thermo Fisher Scientific). Surviving cells were immediately examined to determine protein levels by Immunoprecipitation (IP) and Western blotting.