VRK1 knockdown was performed using three different siRNA specific for VRK1 (Accession number NM_003384 ): siVRK1-01 (siV-01), siVRK1-02 (siV-02), siVRK1-03 (siV-03) and siVRK1-09 (siV-09) (all from Dharmacon RNA Technologies- GE Healthcare). The sequences targeted by these VRK1 siRNA oligonucleotides were siVRK1-01: GAAAGAGAGTCCAGAAGTA; siVRK1-02: CAAGGAACCTGGTGTTGAA; si-VRK1-03: GGAAUGGAAAGUAGGAUUA; and siVRK1-09: AGGUGUACUUGGUAGAUUA. As negative control the “ON-TARGETplus siCONTROL Non-targeting siRNA” (siCt) (Dharmacon) was used. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON) labelled with a red fluorochrome. All of them have been previously used34 (link)48 (link)51 (link).
Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent” (Invitrogen) according to manufacturer instructions. After transfection cells were processed for specific experiments at the times indicated in them. For rescue experiments, cells were transfected with the indicated siRNA using Lipofectamine 2000 Reagent, and 36 hours later, cells were retransfected with plasmids using JetPI reagent (Poly Plus, Ilkirch, France) according to manufacturer instructions. Targeted protein and plasmid expression were analysed thirty-six hours after the second transfection51 (link).
Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent” (Invitrogen) according to manufacturer instructions. After transfection cells were processed for specific experiments at the times indicated in them. For rescue experiments, cells were transfected with the indicated siRNA using Lipofectamine 2000 Reagent, and 36 hours later, cells were retransfected with plasmids using JetPI reagent (Poly Plus, Ilkirch, France) according to manufacturer instructions. Targeted protein and plasmid expression were analysed thirty-six hours after the second transfection51 (link).
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