Latrunculin b sc 203318

HEK-293 T cells, MDA-MB-231 cells and Hela cells were cultured in DMEM medium supplemented with 10% fetal bovine serum in a 5% CO₂ incubator at 37 °C, while T47D cells in 1640 medium. Authentication of the cells was performed by short tandem repeat (STR) analysis in Beijing Microread Genetics Co., Ltd. (Beijing, China). A detailed experimental procedure for primary breast cancer cells was described in the study of Kobayashi et al. [12 (link)]. Latrunculin B (SC-203318) was obtained from Santa Cruz Biotechnology. LY3000328 (MCE-HY-15533) was obtained from MedChemExpress.

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.