For displacement binding assays, A431 cells were grown until confluence on 24-well tissue culture plates (Falcon).
In ligand binding assays, SkBr3 cells were grown in 10-cm cell-culture dishes, washed two times and incubated either with 1 nM [2,4,6,7-3H]E2 (89 Ci/mmol; Amersham Bioscience, GE Healthcare, Milan, Italy) or with 50 nM [5,6-3H] nicotinic acid 50-600 Ci/mmol; BIOTREND, Chemikalien GmbH, Köln, Germany) in the presence or absence of increasing concentrations of nonlabeled competitors, as indicated.
To measure the kinetics of binding of EGF, cells were grown for 24 hours in 6-cm dishes and serum-deprived for 4 to 6 hours at 37°C, followed by a 1-hour incubation on ice with indicated amounts of Rh-EGF.
To visualize NLX binding, A7 and M2 cells were grown on chambered slides (Nalge Nunc, Naperville, IL) to 80% confluency.
In ligand binding assay for GPER, SkBr3 cells were grown in 10-cm cell culture dishes, washed two times and incubated with 1 nM [2,4,6,7-3H]E2 (89 Ci/mmol; Ge Healthcare, Milan, Italy) in the presence or absence of an increasing concentration of nonlabeled competitors (E2, G-1, OHT and MIBE).
Rac1 binding: Primary keratinocytes, HF1 cells and SiHa cells were grown in 10 cm plates to 30% confluence, lysed with a buffer containing 50 mM Tris pH8.0, 0.5 M NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 10 mM MgCl2 and protease inhibitors, sonicated and centrifuged.
Cells were grown in low binding conditions for 14 days and collected for analysis.
If cells were grown in the yeast form, Tpk2 binding occurred most frequently within ORFs at ACCAC, CCACC or CAGC motifs.
Equivalent numbers of cells were grown overnight in 24-well plates and cell surface binding assays were performed as described previously [ 21].
VCaP cells were grown in the presence or absence of the synthetic AR agonist metribolone (R1881) to characterize AR binding in high and low androgen conditions respectively.
