| 5′-TGCGTCAAACTTAGAGGAC-3′ |
The plasmids for retroviral expression were constructed by subcloning the cDNAs encoding cyan fluorescent protein (CFP), or CFP-tagged human SSH1 (CFP-hSSH1), mouse SSH2 (mSSH2-CFP), human wild-type (WT) LIMK1 (hLIMK1(WT)-CFP), or human kinase-dead LIMK1 (hLIMK1(D460A)-CFP) (30), or Myc-tagged chick cofilin (ch-cofilin) or ADF (ch-ADF), into the pLNCX-neo retroviral vector (Clontech, Palo Alto, CA). The siRNA-targeting constructs were generated using the pSUPER.retro.puro vector (Oligo-Engine, Seattle, WA), as described previously (32). The 19-base targeting sequences used in this study were as follows: 5′-GAACTGTCGACCAAGAAAG-3′ (rat SSH1), 5′-TGCGTCAAACTTAGAGGAC-3′ (rat SSH2), 5′-GCTGGAACAATGGCTAGAA-3′ (rat LIMK1), 5′-GCACGAATTACAAGCTAAC-3′ (rat cofilin), and 5′-GCACGAGTATCAAGCAAAT-3′ (rat ADF). As a control, we used a nontargeting sequence, 5′-TCTTCCCCCAAGAAAGATA-3′, which does not exist in the rat genome.To generate retroviral supernatants, Plat-E retrovirus packaging cells (33) were transfected with pLNCX-neo or pSUPER.retro.puro plasmids using FuGENE6 (Roche Applied Science, Mannheim, Germany). At 48 h after transfection, the culture medium was centrifuged, and the viral supernatant was used for infection after the addition of 8 μg/ml polybrene. Infected MM1 cells were cultured for 24 h, washed, and selected by culturing for 48 h with 2 μg/ml puromycin for cells infected with pSUPER.retro.puro retrovirus or for 2 weeks with 250 μg/ml G418 (Nacalai Tesque, Kyoto, Japan) for cells infected with pLNCX retrovirus. |
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