|Use siRNAs concentartion at 100 nM.
Add diluted siRNA to diluted
Reagent (1:1 ratio).
Incubate cells for 48-72 h at 37°C.
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. Full paper
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