These three receptors, i. e., LPA1, LPA2, and LPA3, possess putative phosphorylation sites for a variety of protein kinases, particularly GRKs and PKC isoforms, with marked differences among them [4]. These receptors fused to the enhanced greenPLOS ONE | DOI:10.1371/journal.pone.0140583 October 16,2 /LPA1, LPA2, and LPA3 Phosphorylation and Internalizationfluorescent protein (eGFP) were expressed in C9 cells and their signaling, sensitivity to PKC activation, phosphorylation, and internalization were studied comparatively. Our results Nilotinib site clearly indicate that LPA1, LPA2, and LPA3 receptors are phosphorylated and internalize in response to LPA and PKC activation. Differences in agonist sensitivity and degree of internalization/ desensitization were also observed.Materials and Methods 1. MaterialsLPA (oleyl-sn-glycerol 3-phosphate), phorbol myristate acetate (PMA), G418, Ham’s F12 Kaighn’s modification medium, protease inhibitors and DNA purification kits were purchased from Sigma Chemical Co. Phosphate-free Dulbecco’s modified Eagle’s medium, fetal bovine serum, trypsin, antibiotics, and other reagents used for cell Metformin (hydrochloride)MedChemExpress Metformin (hydrochloride) Culture were from Life Technologies. Fura-2 AM was obtained from Invitrogen and agarose-coupled protein A, from Upstate Biotechnology. Bisindolylmaleimide I and AG1478 were purchased from Calbiochem whereas EGF was obtained from Preprotech. [32P]Pi (8,500?,120 Ci/mmol) was obtained from Perkin Elmer Life Sciences. The plasmid construction for the expression of mouse LPA1 receptor fused to the eGFP was previously described [11] and those used for the expression of human LPA2 and LPA3, also fused to the eGFP at the carboxyl termini, were obtained from GeneCopoeia and OriGene, respectively. Rabbit polyclonal antibodies against ERK 1/2 and phosphoERK 1/2 were from Cell Signaling Technology. Secondary antibodies were obtained from Zymed. For Western blotting an anti-GFP monoclonal antibody from Clontech was employed. Rabbit antisera against eGFP were generated at our laboratory using standard procedures [25] by immunizing New Zealand rabbits with E. coli-overespressed GST-fused eGFP (1 mg/ kg, each 2 weeks for at least 6 times) and have been previously characterized and compared with commercial antibodies [11, 12, 14]. Animals were handled and maintained in individual cages, with free access to water and rabbit chow, in rooms with controlled air temperature and lighting (12 h/12 h); handling and bleeding (marginal ear vein) were performed under the direct supervision of one of the Veterinary Doctors in change of journal.pone.0158910 the animal facility.2. Cell culture and transfectionC9 cells (Clone 9, rat hepatic epithelial cells, CRL-1439TM), obtained directly from American Type Culture Collection, were cultured in Ham’s F12 Kaighn’s modification medium supplemented with 10 fetal bovine serum, 100 g/ml streptomycin, 100 units/ml penicillin and 0.25 g/ml amphotericin B at 37 under a 95 air and 5 CO2 atmosphere, as described previously [11]. The medium was replaced with one containing 1 fetal bovine serum, 12?6 h before the experiment. C9 cells stably expressing the mouse LPA1 receptor fused at the carboxyl termini with eGFP were those previously described [11]. Stable expression of the human LPA2 and LPA3 receptors fused j.neuron.2016.04.018 to eGFP was obtained by transfecting wild type C9 cells with the plasmid constructs described above using lipofectamine 2000, following the manufacturer’s instructions; cells were transfected three times to increase the gene trans.These three receptors, i. e., LPA1, LPA2, and LPA3, possess putative phosphorylation sites for a variety of protein kinases, particularly GRKs and PKC isoforms, with marked differences among them [4]. These receptors fused to the enhanced greenPLOS ONE | DOI:10.1371/journal.pone.0140583 October 16,2 /LPA1, LPA2, and LPA3 Phosphorylation and Internalizationfluorescent protein (eGFP) were expressed in C9 cells and their signaling, sensitivity to PKC activation, phosphorylation, and internalization were studied comparatively. Our results clearly indicate that LPA1, LPA2, and LPA3 receptors are phosphorylated and internalize in response to LPA and PKC activation. Differences in agonist sensitivity and degree of internalization/ desensitization were also observed.Materials and Methods 1. MaterialsLPA (oleyl-sn-glycerol 3-phosphate), phorbol myristate acetate (PMA), G418, Ham’s F12 Kaighn’s modification medium, protease inhibitors and DNA purification kits were purchased from Sigma Chemical Co. Phosphate-free Dulbecco’s modified Eagle’s medium, fetal bovine serum, trypsin, antibiotics, and other reagents used for cell culture were from Life Technologies. Fura-2 AM was obtained from Invitrogen and agarose-coupled protein A, from Upstate Biotechnology. Bisindolylmaleimide I and AG1478 were purchased from Calbiochem whereas EGF was obtained from Preprotech. [32P]Pi (8,500?,120 Ci/mmol) was obtained from Perkin Elmer Life Sciences. The plasmid construction for the expression of mouse LPA1 receptor fused to the eGFP was previously described [11] and those used for the expression of human LPA2 and LPA3, also fused to the eGFP at the carboxyl termini, were obtained from GeneCopoeia and OriGene, respectively. Rabbit polyclonal antibodies against ERK 1/2 and phosphoERK 1/2 were from Cell Signaling Technology. Secondary antibodies were obtained from Zymed. For Western blotting an anti-GFP monoclonal antibody from Clontech was employed. Rabbit antisera against eGFP were generated at our laboratory using standard procedures [25] by immunizing New Zealand rabbits with E. coli-overespressed GST-fused eGFP (1 mg/ kg, each 2 weeks for at least 6 times) and have been previously characterized and compared with commercial antibodies [11, 12, 14]. Animals were handled and maintained in individual cages, with free access to water and rabbit chow, in rooms with controlled air temperature and lighting (12 h/12 h); handling and bleeding (marginal ear vein) were performed under the direct supervision of one of the Veterinary Doctors in change of journal.pone.0158910 the animal facility.2. Cell culture and transfectionC9 cells (Clone 9, rat hepatic epithelial cells, CRL-1439TM), obtained directly from American Type Culture Collection, were cultured in Ham’s F12 Kaighn’s modification medium supplemented with 10 fetal bovine serum, 100 g/ml streptomycin, 100 units/ml penicillin and 0.25 g/ml amphotericin B at 37 under a 95 air and 5 CO2 atmosphere, as described previously [11]. The medium was replaced with one containing 1 fetal bovine serum, 12?6 h before the experiment. C9 cells stably expressing the mouse LPA1 receptor fused at the carboxyl termini with eGFP were those previously described [11]. Stable expression of the human LPA2 and LPA3 receptors fused j.neuron.2016.04.018 to eGFP was obtained by transfecting wild type C9 cells with the plasmid constructs described above using lipofectamine 2000, following the manufacturer’s instructions; cells were transfected three times to increase the gene trans.
