The human CHMP5-shRNA (5’-CCGGGAGTTGGATGCACTAGGTGATCTCGAGATCACCTAGTGCATCCAACTCTTTTTTG-3’), human CHMP6-shRNA (5’-CCGGGCGCAATCACTCAGGAACAAACTCGAGTTTGTTCCTGAGTGATTGCGCTTTTTTG-3’), and control empty shRNA (pLKO.1) were obtained from Sigma-Aldrich (St. Louis, MO, USA). RNAi was performed using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Stable knockdown cells were selected by adding puromycin.
Control empty shrna plko 1
Manufactured by Merck Group
Sourced in United States
The pLKO.1 plasmid is a lentiviral vector used as a control in shRNA experiments. It contains a puromycin resistance gene for selection and a cloning site for insertion of shRNA sequences. The vector itself does not express any shRNA, making it a suitable control for experiments involving shRNA knockdown.
Automatically generated - may contain errors
2 protocols using control empty shrna plko 1
1
Stable Knockdown of CHMP5 and CHMP6
2
Targeted Knockdown of Autophagy Genes
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The predesigned human ATG5 shRNA-1 (TRCN0000330394), ATG5 shRNA-2 (TRCN0000151474), ATG7 shRNA-1 (TRCN0000007584), ATG7 shRNA-2 (TRCN0000364479), ACSL4 shRNA-1 (TRCN0000045541), ACSL4 shRNA-2 (TRCN0000045539), NCOA4 shRNA (TRCN0000236186), and control empty shRNA (pLKO.1) in a lentiviral format were obtained from Sigma-Aldrich. We seeded 1 × 105 cells in each well of a 12-well plate in 500 μl of complete medium and transduced them by lentiviral vectors at a multiplicity of infection of 10:1. Transduction was carried out in the presence of polybrene (8 μg/ml; Thermo Fisher Scientific, TR1003G) in an antibiotic-free medium. After recovering with complete culture medium, puromycin (Thermo Fisher Scientific, A1113802, 5 μg/ml) was used for the selection of transduced cells. The efficiency of RNAi was checked by western blot analysis of target proteins.
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