Additional file 6: of Genetic variants in the upstream region of activin receptor IIA are associated with female fertility in Japanese Black cattle

Expression of pCAGGS- ACVR2A in LβT2 cells. (A) To determine whether the pCAGGS-ACVR2A plasmid was expressed in LβT2 cells, we transfected 2 × 105 cells per well in a 24-well plate with a mixture of 200 ng of the FSHB promoter-reporter plasmid, pCAGGS-ACVR2A (the amount of each plasmid is indicated in the lanes) and 10 ng of pRL-TK Renilla. HA-ACVR2A expression was confirmed by western blot analysis using an anti-HA antibody. Unstained Precision Plus protein standards were used as a marker (BioRad, Cat. #161-0363). HA-ACVR2A expression was detected with multiple bands (~57.8 kDa) because TGF-beta type-II receptors are normally modified by phosphorylation and glycosylation, causing then to migrate heterogeneously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. (B) Relative ACVR2A band intensities were measured using the ImageQuant TL Analysis Toolbox. The amounts of loaded proteins were calibrated by Coomassie Brilliant Blue-stained bands between 37 and 75 kDa. The bars represent the mean ± SEM observed in triplicate from 3 independent experiments. (PPTX 706 kb)