Cmv mcs ha sv40 neomycin

The following plasmids were purchased from GeneChem (Shanghai, China): lentivirus plasmid (Ubi-MCS-3FLAG-SV40-puromycin) carrying the full-length human STAMBP CDS (gene ID: 10617) and lentivirus plasmid (CMV-MCS-HA-SV40-Neomycin) carrying the full-length human RAI14 CDS (gene ID: 26064). Cells were plated at a density of 3 × 10⁵ cells/well and cultured for 24 h. Then, lentivirus was added to the cells at a multiplicity of infection of 10. After transfection for 48 h, 1 µg/ml puromycin (Selleck Chem, S7417) and 2 mg/ml G418 (Sigma, 108321-42-2) were used to select for cells with successfully transfected STAMBP and RAI14 plasmids, respectively.

The plasmids and lentivirus transfection assays were used to upregulate the expression levels of the contained gene. For plasmid transfection, the full length of human USP14 CDS plasmids (CMV-MCS-Flag-SV40-neomycin), the full length of human USP22 CDS plasmids (CMV-MCS-6His-SV40-neomycin), the full length of human AR-V7 CDS plasmids (CMV-MCS-HA-SV40-neomycin), and their control plasmids were constructed and purchased from GeneChem (Shanghai, China). Mix Lipofectamine 3000 (Invitrogen) (2 μl P3000 and 2 μl Lipofectamine 3000) and 1 μg plasmids in 500 μl RPMI opti-MEM (Gibco) were added into the cells that have been preinoculated and cultured in dishes for 24 h with a final concentration of the plasmids at 0.75 μg/ml.Cells were treated for 48 h for other analysis.
For lentivirus transfection, lentivirus (Ubi-MCS-HA-SV40-puromycin) containing the full length of human AR-V7 CDS and its control lentivirus were also constructed and purchased from GeneChem (Shanghai, China). After the cells were seeded in dishes or plates and cultured for 24 h, the fresh medium containing 5 μg/ml polybrene(Santa Cruz, CA) and lentivirus at a multiplicity of infection of 10 was used to culture for 48 h. Puromycin at a concentration of 2 μg/ml was used to select and retain the successfully infected cells.