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A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

Figure 2

Immunoblot of total membranes under reduced and non-reduced conditions from stably transfected cells:

CHO cells stably expressing GPI-TSHR-ecd were treated with 0, 10 and 1000 µU/ml of bovine TSH for 1 hr at 37°C. Membranes prepared from untreated and treated cells was divided into 30 ug/lane and treated with 5X sample buffer containing reducing agent (+DTT: 100 mM final) and non-reducing agent (-DTT) and separated on a 12% SDS-PAGE. Proteins were transferred onto PVDF and probed by 2 ug/ml TSHR antibody M4 (epitope 322–341) followed by detection with anti-mouse HRP (1:20,000) and developed using pico SuperSignal ECL (Pierce Ltd). Panel A) Shows the non-reduced untreated and treated samples. Oligomeric forms of 200–250 KD were observed in addition to the mature 100KD and less mature 75 KD monomeric forms GPI linked TSHRs. Panel B) Untreated and treated samples after reduction with DTT for 45 minutes at 50°C showing no higher order forms of the TSHR-ecd indicating the instability of the TSHR-ecd higher order forms. This is a representative blot from two experiments performed under identical conditions. Panel C) the specificity of the antibody M4 used for immunoblotting throughout the study is indicated in this blot. Lysates from untransfected HEK 293 cells and TSHRGFP transfected wild type cells were resolved on SDS-PAGE and immunblotted with 2 ug/ml of M4 antibody. M4 did not have any reactivity with untransfected lysate as opposed to TSHR transfected lysate where it detected the monomeric and multimeric forms of the receptor.

Figure 2

doi: https://doi.org/10.1371/journal.pone.0009449.g002