Lysophosphatidic acid lpa sc 201053

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.

Verteporfin (VP) (SML05434), MG132 (C2211), 5Z-7-Oxozeaenol (O9890), and IL-1β (SRP6169 (human) and SRP8033(mouse)) were purchased from Sigma. SP600125 (T3109) was purchased from TargetMol. Lysophosphatidic acid (LPA) (sc-201053) was purchased from Santa Cruz. Cycloheximide (CHX) (2112 s) and TNFα (5178 (mouse) and 8902 (human)) was purchased from Cell Signaling. The Phos-tag TM Acrylamide AAL-107 was purchased from the NARD Institute. Kinase-dead TAK1 (TAK1-KD) construct was created by mutating lysine 63 to tryptophan. Kinase-dead IKKα (IKKα-KD) construct was created by mutating lysine 44 to alanine. Kinase-dead IKKβ (IKKβ-KD) construct was created by mutating lysine 44 to methionine.