ELISA binding assays with PRLR ECD were performed as previously described, except 0.25 μg/ml of ECD protein was bound to plates [24].

For cellular binding studies, cells were harvested and sorted as previously described [24] using anti-PRLR (mouse version of h16f) or control antibody ((−)Co). When necessary to dissociate cell aggregates, cells were briefly treated with Accutase (Millipore, #SCR005). Data was analyzed using Becton Dickinson FACSDIVA software. For quantitative determination of PRLR on the cell surface, QIFIKIT (Dako) was carried out according to the manufacturer’s instructions, as previously described [25].

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