VRK1 Silencing by siRNA Protocol

The suppression of VRK1 expression was done using specific siRNA from Dharmacon RNA Technologies (Dharmacon, Inc.; Lafayette, CO, USA) or OriGene Technologies (Rockville, MD, USA), respectively. Specific silencing of VRK1 was performed using two different siRNA: siVRK1-02 (siV1-02) and siVRK1-03 (siV1-03) from Dharmacon (DHARMACON RNA Technologies). The sequence target of the two VRK1 siRNA oligos was siVRK1-02: CAAGGAACCTGGTGTTGAA and siVRK1-03: GGAAUGGAAAGUAGGAUUA. As negative control, indicated as siCt in experiments, the “ON-TARGETplus siCONTROL Non-targeting siRNA” from DHARMACON was used. The efficiency of RNAi transfection was determined with “siGLO RISC-free siRNA” (DHARMACON). Briefly, cells were transfected with the indicated siRNA at a concentration of 20 nM using Lipofectamine 2000 Reagent (Invitrogen) according to the manufacturer’s instructions. After transfection, cells were processed at the times indicated in specific experiment that were performed as previously reported [28 (link), 45 (link), 47 (link), 67 (link)].