The next morning, media was replaced with intoxication media containing 5 µg/ml acridine orange (Calbiochem, cat# 113000) for 15 min under otherwise standard culture conditions.
The next morning, media were replaced with 2 ml media containing 125 ng/ml TBG-dysprosium (Dy).
The next morning the ten colonies were grown in 1 ml LB media containing the appropriate antibiotic.
The culture media containing lentivirus was harvested twice every 24 hours and filtered, and polybrene was added at 8 µg/mL.
In the morning, each strain was diluted 1 2000 into 200 μL of the same media containing 0 nM, 100 nM, 1 μM, or 10 μM of β-estradiol in a well of a 96-well flat-bottom plate (Costar).
Forty-eight hours post transfection, the media containing the virus was collected, filtered, and centrifuged at 70,952 × g for 2 h at 4 °C.
Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 media containing L-glutamine 300 g/L (Gibco/Invitrogen, Paisley, UK), supplemented with 5% fetal calf serum (Labtech/Biosera, Sussex, UK) referred to as R5 media.
Equal volumes of media containing AP or AP-tagged proteins were pre-cleared with Protein A-Agarose beads at 4°C for 2 h and then incubated with the LRP4ecto-bead conjugates at 4°C overnight.
Yeast cells were grown overnight in media containing L-proline as nitrogen source instead of ammonium sulfate.
After 48 h in culture, media was removed, and phenol red-free media containing L-NAME at different concentrations was added to the wells.
Deletion of lxr3 led to a significant reduction in NADPH specific LXR activity after replacement to both media containing l-arabinose, while NADH specific LXR activity remained constant (e.g., 0.6 nkat/mg on l-arabinose-containing minimal medium).
