Differential recognition by murine TSHR mAb of the wild-type TSHR ectodomain and the GPI-anchored ectodomain. Flow cytometry was performed with the following murine mAb’s: (a) mAb 3BD10, epitope including the cysteine-rich segment at the extreme N terminus of the TSHR ectodomain; (b) mAb 2C11, epitope in the C peptide region of the TSHR ectodomain, present only in the uncleaved, but not in the cleaved, TSHR; (c) normal mouse serum (NMS) included as a control. The assays used the following cell lines: TSHR-10,000, ECD-GPI, and CHO. TSHR-10,000 is the CHO cell line stably expressing the wild-type TSH holoreceptor. The transgenome has been amplified using the dihydrofolate reductase minigene approach (adaptation to final methotrexate concentration of 10,000 nM), leading to very high TSHR expression on the cell surface (∼2 × 10⁶/cell) (30). ECD-GPI is the CHO cell line stably expressing the TSHR ectodomain anchored to the plasma membrane by a GPI moiety. CHO are untransfected CHO cells. Note the approximately 100-fold difference in 3BD10 versus 2C11 recognition of the TSHR holoreceptor, despite their similar recognition of the ECD-GPI cell line.