pSUPER.retro.puro

Protocol tips

Upstream tips	Protocol tips	Downstream tips
5′-GAACTGTCGACCAAGAAAG-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.

Upstream tips
5′-GAACTGTCGACCAAGAAAG-3′
Protocol tips
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.

Publication protocol

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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