To evaluate the effect of P1_WT to inhibit RHEB-mTORΔN interaction,
we used the AlphaLISA-based assay as previously described.12 (link),20 (link) First, we evaluated RHEB-mTORΔN interaction by
mixing different concentrations of 6xHis-mTORΔN with
1 μM biotinylated RHEB in a 384-well OptiPlateTM (PerkinElmer,
USA) followed by adding 100 μg/mL streptavidin-coated donor
beads and 200 μg/mL anti-6xHis-coated acceptor beads. The plate
was then sealed, covered, and incubated in the dark for >1 h at
room
temperature. The alpha signal was then measured by an EnSpire plate
reader.
The effect ofP1_WT on RHEB-mTORΔN interaction was evaluated using the same method with some modifications.
Increasing concentrations ofP1_WT were incubated with
1 μM biotinylated RHEB; then, 1 μM of 6xHis-mTORΔN was added followed by 100 μg/mL of streptavidin-coated donor
beads and 200 μg/mL of anti-6xHis-coated acceptor beads. The
plate was then sealed, covered, and incubated in the dark for >1
h
at room temperature. The alpha signal was then measured by an EnSpire
plate reader.
we used the AlphaLISA-based assay as previously described.12 (link),20 (link) First, we evaluated RHEB-mTORΔN interaction by
mixing different concentrations of 6xHis-mTORΔN with
1 μM biotinylated RHEB in a 384-well OptiPlateTM (PerkinElmer,
USA) followed by adding 100 μg/mL streptavidin-coated donor
beads and 200 μg/mL anti-6xHis-coated acceptor beads. The plate
was then sealed, covered, and incubated in the dark for >1 h at
room
temperature. The alpha signal was then measured by an EnSpire plate
reader.
The effect of
Increasing concentrations of
1 μM biotinylated RHEB; then, 1 μM of 6xHis-mTORΔN was added followed by 100 μg/mL of streptavidin-coated donor
beads and 200 μg/mL of anti-6xHis-coated acceptor beads. The
plate was then sealed, covered, and incubated in the dark for >1
h
at room temperature. The alpha signal was then measured by an EnSpire
plate reader.
