siGENOME Human PAK1 siRNA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Use siRNAs concentartion at 100 nM. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 48-72 h at 37°C.

Protocol tips
Use siRNAs concentartion at 100 nM. Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio). Incubate cells for 48-72 h at 37°C.

Publication protocol

HeLa cells were cultured at 37°C in a humidified 5% CO2 atmosphere in Eagle’s Minimum Essential Medium (EMEM) (Sigma) containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin. RNA interference-mediated gene knockdown was performed using validated Qiagen HP small-interfering RNAs (siRNAs) for clathrin (SI299880), caveolin1 (SI00299642) and caveolin2 (SI02664389). Dynamin I/II siRNA (sc43736) was from Santa Cruz Biotechnology. PKCδ siRNAs (Llado et al., 2004) were designed and validated as described previously. PAK1 siRNAs specific were purchased from Dharmacon (M-003521-04). Negative-control (NC) siRNAs were purchased from Sigma Aldrich or Dharmacon. HeLa cells were transfected with 100 nM of the indicated siRNAs for 48–72 h using LipofectamineTM RNAiMax transfection reagent (Invitrogen) according to the manufacturer’s protocol. Transfection efficiency and effect were evaluated by Western blotting using the indicated antibodies.

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Manufacturer protocol

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