Transfection-based RAC1B knockdown

On day 1, cells were seeded into Nunclon^TM Delta Surface plates (Nunc, Roskilde, Denmark), and transfected twice, on days 2 and 3, serum-free with either 50 nM of prevalidated siRNAs specific for RAC1B [18 (link)] or a scrambled control for 4 h using Lipofectamine 2000 (Panc1 cells) or Lipofectamine RNAiMAX (Capan1 and Colo357 cells) (both from Life Technologies). The procedure for plasmid vectors encoding HA-RAC1B (in β) or MYC-RAC1-L61 (in pRK5) was identical except that cells were transfected only once Afterwards, cells received normal growth medium and were incubated for another 24 or 48 h prior to qPCR, immunoblot analysis, or real-time migration assay.