ON-TARGETplus Human PAK1 (5058) siRNA - SMARTpool

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	"Caco-2 cells were treated with small interfering RNA (siRNA)using a modification of our previously published methods (Forsyth et al., 2010). Briefly, 105 cells are combined with 11 picomoles of siRNA in 50 ll Lipofectamine (Invitrogen) and 50 ul Optimem (Invitrogen), mixed by gently shaking and then plated on Type 1 collagencoated Corning 12 mm PET culture inserts (#3460) with addition of another 400 ul (upper well) and 1 ml (lower well) Optimem. Inserts are used for permeability studies 72 to 96 hours after plating, when the cells are confluent. PAK1 and iNOS siRNA were On Target Smartpool from Dharmacon (Lafayette, CO), whereas control (nontargeting) siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA)"

Protocol tips

"Caco-2 cells were treated with small interfering RNA (siRNA)using a modification of our previously published methods (Forsyth et al., 2010). Briefly, 105 cells are combined with 11 picomoles of siRNA in 50 ll Lipofectamine (Invitrogen) and 50 ul Optimem (Invitrogen), mixed by gently shaking and then plated on Type 1 collagencoated Corning 12 mm PET culture inserts (#3460) with addition of another 400 ul (upper well) and 1 ml (lower well) Optimem. Inserts are used for permeability studies 72 to 96 hours after plating, when the cells are confluent. PAK1 and iNOS siRNA were On Target Smartpool from Dharmacon (Lafayette, CO), whereas control (nontargeting) siRNA was from Santa Cruz Biotechnology (Santa Cruz, CA)"

Publication protocol

Caco-2 cells were treated with small interfering RNA (siRNA)using a modification of our previously published methods (Forsyth
et al., 2010). Briefly, 105 cells are combined with 11 picomoles of siRNA in 50 ll Lipofectamine (Invitrogen) and 50 ul Optimem (Invitrogen), mixed by gently shaking and then plated on Type 1 collagencoated Corning 12 mm PET culture inserts (#3460) with addition of another 400 ul (upper well) and 1 ml (lower well) Optimem. Inserts are used for permeability studies 72 to 96 hours after plating, when
the cells are confluent. PAK1 and iNOS siRNA were On Target Smartpool from Dharmacon (Lafayette, CO), whereas control (nontargeting) siRNA was from Santa Cruz Biotechnology (Santa
Cruz, CA)

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