Degradation of human androgen receptor monitored by HTRF and western blot in cells

A comparison between the ARCC-4 PROTAC compound dose-dependent degradation of Androgen receptors monitored by Western Blot and HTRF Human Androgen Receptor kit was done. The human prostate cancer cell line LNCaP was seeded in a 96-well culture-treated plate under 75,000 cells / well in complete culture medium, and incubated overnight at 37 ° C, 5% CO2. The cells were treated with the ARCC-4 PROTAC compound to a concentration range of 5nM to 10 000 nM for 24h. After treatment, the cells were lyzed with 50 µL of supplemented lysis buffer # 3 for 30 minutes at RT under gentle shaking. For the detection step with the Human Androgen Receptor kit, 16 µL of cell lysate were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Human Androgen Receptor detection reagents were added. The HTRF signal was recorded after 2h of incubation. The HTRF Human Androgen receptor kit enables the determination of a DC50 (concentration at which 50% of the target protein has been degraded) of 900 nM for the ARCC-4 PROTAC compound to degrade Human Androgen receptors in LNCap cells. The degradation of both AR-WT and AR-V7 forms expressed in LNCaP cells by the ARCC-4 PROTAC compound was evaluated by Western Blot. Results demonstrate that only the AR-WT is degraded by the ARCC-4 PROTAC compound, explaining the partial degradation monitored by the HTRF Human Androgen receptor kit which can detect both forms. The DC50 value for the ARCC-4 PROTAC compound to degrade Human Androgen receptors in LNCap cells are consistent both with the HTRF Human Androgen receptor kit and Western Blot.

 

HTRF Human androgen receptor assay compared to western blot

Human breast cancer cells were cultured in a T175 flask in a complete culture medium for 48h at 37 ° C, 5% CO2. The cells were lyzed with 3 mL of supplemented lysis buffer # 3 for 30 minutes at RT under gentle shaking. Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Human Androgen Receptor detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot. Using the HTRF Human Androgen Receptor assay, 1,500 cells / well were enough to detect a significant signal, while 6,500 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, in these conditions, the HTRF Human Androgen Receptor assay was 4.5 times more sensitive than the Western Blot technique.