TSHR ectodomain linked to GPI as a source of antigen.

<p><b>Panel A -</b> The source of the native antigen for epitope protection studies was the entire TSHR ectodomain (ECD) protein which was anchored to the plasma membrane via a GPI linker. This link could be cleaved by phospholipase C treatment to obtain large amounts of purified receptor ectodomain. <b>Panel B -</b> Surface expression of the TSHR-GPI construct on CHO cells is illustrated by staining with TSHR-mAb M1 directly conjugated to Alexa 488. <b>Panel C</b> - Immunoprecipitation of solubilized receptors from total membrane preparations obtained from GPI-TSHR-ECD cells precipitated using MS-1 (2 ug/ml) (lane 1) and probed with an antibody to the N-terminus of the receptor (A10). A specific band corresponding to 74KD protein was observed in lane 1 whereas no immunoprecipitation was observed with MS-1 using membranes prepared from untransfected CHO cells (lane 2) and probed with an antibody directed to amino terminus of receptor (A10, residues 22–41). <b>Panel D -</b> PI-PLC digest obtained from GPI-TSHR-ECD cells immunoprecipitated with MS-1 (2 ug/ml) (lane 2) and from GPI-TSHR-ECD cells untreated with PI-PLC (lane 1). A specific band corresponding to a 74KD protein was observed in lane 2 compared to a faint lower weight band obtained with vehicle alone (lane 1). The faint band maybe is the result of CHO-TSHR cell breakdown that resulted in some binding of TSHR-mAb.