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

<p>HEK 293 cells transiently transfected with HA-GPI-TSHR-ecd were treated with 0, 10 and 1000 µU/ml of bovine TSH for 1 hr at 37°C. Total membranes prepared from untreated and treated cells were divided into 30 µg/lane and treated with 5X sample buffer containing reducing agent (+DTT:100 mM DTT final) and non-reducing agent (-DTT) and the proteins separated by 12% SDS-PAGE. The blots were probed further using 2 µg/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). <b>Panel A</b>) Shows the untreated and TSH treated samples under non-reduced conditions. <b>Panel B</b>) The same set of samples as in Panel A after reduction with DTT for 45minutes at 50°C. Results are similar to that observed with stably expressed cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009449#pone-0009449-g002" target="_blank">Figure 2</a>. <b>Panel C</b>) Flow cytometric data of untreated versus treated cells. TSH treatment did not alter the expression profile of HA in these HA-GPI-TSHR-ecd transiently transfected cells. The expression levels of the tagged receptor are indicated by their mean fluorescence intensity (MFI).