Polythylenimine pei

HEK293A (ATCC), HEK293T (ATCC), and HeLa (ATCC) cells were cultured in DMEM medium with 10% FBS. All the cells were cultured at 37 °C in cell culture incubator with humidified environment in 5% CO₂. Lipofectamine 3000 (Invitrogen) or polythylenimine (PEI) (Polysciences) transfection reagents were used for plasmid transfection. All cell lines used were tested for mycoplasma contamination using the Mycoplasma Test Kit (Shanghai Yise Medical Technology Co. Ltd, PM008).

HEK293A, HEK293T, NIH3T3, and A549 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. All the cells were cultured at 37 °C in cell culture incubator with humidified environment in 5% CO₂. Lipofectamine 3000 (Invitrogen) or polythylenimine (PEI) (Polysciences) transfection reagents were used for plasmid transfection. TNF and FGF2 were purchased from Peprotech. HEK293A (3 × 10⁵/well), and NIH3T3 (1 × 10⁵/well) cells were sparsely seeded on six-well plates and treated with 5 ng/ml TNF or 50 ng/ml FGF2 for different times. MG132 (C2211) was purchased from TargetMol. Lysophosphatidic Acid (LPA) (sc-201053) and Latrunculin B (sc-203318) were purchased from Santa Cruz. Cells were treated with Latrunculin B (200 ng/ml) to disrupt the actin cytoskeleton. We generated a kinase-dead mutant MEKK3-KD, which contains S526/T530 to Ala point mutations, and a kinase-dead mutant MEKK2-KD, which contains S521/T523 to Ala point mutations. The rabbit polyclonal antibody against the phosphorylated of YAP-S371 was produced using the synthetic phosphorylated peptides PGM-S(p)-QELRTMT-C as antigen and purified on a phosphopeptide column.