Functional testing of grass carp activin A and B in GH3 cells.

Abstract

<p>(A) Validation of actvin responsiveness in GH3 cells with pAR3-Lux transfection. GH3 cells were transfected with the activin-responsive pAR3-Lux reporter and challenged for 24 hr with human activin A (10 ng/ml) and B (10 ng/ml), respectively. To confirm that the effects observed were specific for activin, parallel experiments were conducted with activin A and B induction in the presence of human follistatin (300 ng/ml). (B) Effects of carp activin A and B on pAR3-Lux reporter activity expressed in GH3 cells. Conditioned media obtained from CHO cells transfected with the expression vectors for carp activin βA (gc.Act βA) and βB (gc.Act βAB) were used as the source of carp activin A and B, respectively. For functional testing of carp activins, GH3 cells with pAR3-Lux transfection were treated for 24 hr with the conditioned media containing activin A and B, respectively, with parallel treatment of conditioned medium harvested from CHO cells transfected with the blank vector pcDNA3.1(+) as the control. To confirm that the effects on pAR3-Lux reporter activity were indeed mediated by activin, parallel studies with the respective conditioned media were also repeated with co-treatment of follistatin (300 ng/ml). In these experiments, cell lysate was prepared from GH3 cells after drug treatment and used for luciferase (LUC) activity measurement using a Dual-Glo luciferase assay. Data presented are expressed as Mean ± SEM (N = 4) and the groups denoted by different letters represent a significant difference at P < 0.05 (ANOVA followed by Newman-Keuls test).</p

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