Recombinant human dkk 1

The cell lines Huh-7 (Japanese Collection of Research Bioresources, JCRB-0403) and HepG2 (American Type Culture Collection, ATCC-HB-8065) were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Gaithersburg, MD, USA) supplemented with 10% FBS (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA), 100 U/ml penicillin, 100 mg/ml streptomycin at 37℃ in a humidified 5% CO₂ atmosphere. A stable Huh-7 cell line, which overexpresses FLAG-tagged 14-3-3ε, was previously established and maintained in DMEM with 10% FBS and 200 μg/ml G418^{36 (link)}. All pharmacological inhibitors were purchased from Sigma–Aldrich (St. Louis, MO, USA). Recombinant human DKK-1 was purchased from PeproTech (Rocky Hill, NJ, USA).

Zoledronic acid was provided by Novartis (Basel, Switzerland), and atorvastatin, mevalonate, geranyl-geranyl-pyrophosphate (GGPP), farnesyl pyrophosphate (FPP), GGTI-298 and FTI-277 were obtained from Sigma-Aldrich (Munich, Germany). Rac1 inhibitors #1 (553502) and #2 (553511), Y-27632, rho kinase inhibitor (H-1152P) and Clostridium difficile toxin A were from Merck Chemicals (Darmstadt, Germany). Cdc42 inhibitor ML-141 was from Tocris Bioscience (Bristol, UK). Rho inhibitor #2 (BML-EI394) was obtained from Enzo (Lörrach, Germany). Rho inhibitor #1 (CT04) and Rho/Rac/Cdc42 activator I were from Cytoskeleton Inc. (Denver, CO, USA). Recombinant human DKK-1 was obtained from Peprotech (Hamburg, Germany). Antibody for RAP1A (sc-1482) was from Santa Cruz (Heidelberg, Germany), RAS (610001) antibody was from BD Biosciences (Heidelberg, Germany), the DKK-1 (MAB10962) antibody was from R&D Systems (Wiesbaden, Germany) and all other antibodies were obtained from Cell Signaling Technology (Frankfurt, Germany). DKK-1 small interfering RNA (siRNA; s22721 and s22723), Cdc42 siRNAs and nontarget siRNA were purchased from Applied Biosystems (Darmstadt, Germany). Cells were transfected using Dharmafect (Thermo Scientific, Waltham, Massachusetts (MA) USA).

St. 25 embryos were injected in the chorionic membrane with 5–10 nl of recombinant human DKK1 (3 ng) (Peprotech; Cat#120-30-10UG), IWP-2 (12 ng) (Sigma; Cat#686770-61-6) or recombinant human WNT3A (2 ng; Cat#5036-WN-010). For WNT3A co-injection 2 ng DKK1 was used. As controls, 0.1% BSA in PBS or (0.1% BSA, 0.1 mM EDTA (Sigma), 0.5% (w/v) CHAPS (MP Biomedicals), 0.5% (w/v) DMSO) in PBS were used, respectively. After 2 h incubation, embryos were manually hatched and fixed in 4% PFA for immunostaining.