Sc 51907

Recombinant human ATX (also known as Ectonucleotide Pyrophosphatase/Phosphodiesterase-2) was purchased from R&D Systems, Inc., Minneapolis, MN, USA. 1-Oleoyl lysophosphatidic acid sodium salt was from Tocris Bioscience (Bristol, UK). 17-β estradiol (estrogen) was from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). PD98059 [the phosphorylated extracellular signal-regulated kinase (p-ERK) inhibitor] was from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). ATX antibody for western blotting was from Abcam (Cambridge, UK; ab77104) and for immunohistochemistry from Santa Cruz Biotechnology (Texas, USA; sc-374222). Antibodies against LPA receptors (LPA1, LPA2, LPA3) were from Abcam (ab166903, ab38322 and ab219267, respectively). Total/phosphorylated extracellular signal-regulated kinases (t/p-ERK) were from Cell Signaling Technology, Inc. Danvers, MA, USA (4695s and 4370s). Antibodies against GAPDH were from Santa Cruz Biotechnology (sc-51907). Crystal violet staining solution was from Tiangen Biotech Co., Ltd. (Beijing, China). Cell Counting kit (CCK)-8 was from Beyotime Institute of Biotechnology, Haimen, China (C0037). Transwell culture plates were from Corning Incorporated (Corning, NY, USA). Lipofectamine 2000™ transfection reagent was from Invitrogen, Thermo Fisher Scientific, Inc.

Cultured cells were collected and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology). An enhanced BCA protein assay kit was used for total protein quantification. Equal amounts of total protein were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis on 10% gels and transferred to polyvinylidene fluoride membranes. After 2 h of blocking with 5% nonfat powdered milk, the membranes were incubated with primary antibodies against KLF12 (1:1,000 dilution; sc-134,373; Santa Cruz Biotechnology, CA, USA) or GAPDH (1:1,000 dilution; sc-51907; Santa Cruz Biotechnology) overnight at 4°C. The membranes were further incubated for 2 h with a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000 dilution; sc-516102; Santa Cruz Biotechnology) at room temperature, and the protein bands were detected using Thermo Scientific Pierce ECL Plus Substrate (Pierce; Thermo Fisher Scientific, Inc.). GAPDH served as an internal control for KLF12 expression.