Cdc42

MDA-MB-231/-468 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma, #P8340; #P0044). After centrifugation, proteins were recovered in the supernatant. Protein concentration was determined by the Bradford protein assay (Bio-Rad, #5000006). Proteins were separated on SDS-PAGE transferred to PVDF membranes. Primary antibodies PLD2 (1:1000; Cell Signaling, #13904) and Cdc42 (1:500; Invitrogen, #PA1-092) were used. HRP-conjugated anti-rabbit IgG (1:2000: Cell Signaling, #7074) was used as a secondary antibody. Signals were revealed using the ECL substrate (Millipore, #WBKLS0100). To normalize and verify protein amounts equally, membranes were finally stained with amido black solution (0.25% amido black, 45% MeOH, 45% ddH2O, 10% glacial HOAc) and de-stained with the same solution without dye.

The specificities of the siRNA pools were confirmed with the following independent siRNAs: RhoA, 5′-CACAGUGUUUGAGAACUAUTT-3′ and 5′-GCUAGACGUGGGAAGAAAATT-3′; Rac1, 5′-GGAACUAAACUUGAUCUUATT-3′ and 5′-CCUUUGUACGCUUUGCUCATT-3′; Cdc42, 5′-UGAGAUAACUCACCACUGUTT-3′ and 5′-AGAUCUAGUUUAGAAAACATT-3′ (Invitrogen). siGLO RNA-induced silencing complex-free control siRNA was purchased from Thermo Fisher Scientific. In total, 50 nM siRNA was transfected into huESCs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were then incubated in K-SFM for 24 h before further experimentation.