Falcone M, Miyamoto T, Fierro-Renoy F, Macchia E, DeGroot L J
Department of Medicine, University of Chicago, Illinois 60637.
Endocrinology. 1992 Nov;131(5):2419-29. doi: 10.1210/endo.131.5.1425440.
We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform. The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E. coli and their specificity was confirmed by competition studies and immunohistochemistry. Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E. coli. No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E. coli. By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined. However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis. We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis. In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.
我们鉴定了四种能够特异性识别每种甲状腺激素受体（TR）亚型的抗肽多克隆抗体（抗体）。这些抗体免疫沉淀了体外合成的受体以及在大肠杆菌中表达的受体，并且通过竞争研究和免疫组织化学证实了它们的特异性。用免疫原性肽或在大肠杆菌中表达的特异性受体蛋白预先吸附每种抗体后，通过酶联免疫吸附测定法测量的抗体活性降低。在酶联免疫吸附测定中未检测到特异性活性，亲和柱免疫吸附后未观察到核染色，并且在与在大肠杆菌中表达的特异性TR亚型预先吸附后，蛋白质印迹分析中获得的特异性条带消失。通过免疫组织化学研究，我们在大多数检测的组织中检测到了每种受体亚型的协同表达。然而，在心脏和肌肉中，尽管先前通过Northern印迹分析证明了TRβmRNA水平显著，但与α亚型相比，β亚型的表达水平非常低。我们还证明了大鼠睾丸中α和β亚型的不同分布模式。在该组织中，TRα在精原细胞核中显著表达，但在精子细胞中β亚型占主导地位，并且在成熟精子中仅可检测到TRβ。
