Alphascreen surefire pstat5 or pakt assays

AlphaScreen assays were performed as previously described (Newey et al. 2013 (link), Gorvin et al. 2018b (link)). Cells were transiently transfected in 48-well plates with 200 ng of either WT or variant PRLR vectors. After 30 h, cells were incubated in serum-free media for 12 h prior to treatment with human recombinant PRL (PromoCell, Heidelberg, Germany) for 20 min at concentrations ranging from 0 to 1000 ng/mL. Cells were lysed in Surefire lysis buffer, and AlphaScreen Surefire pSTAT5 or pAkt assays (PerkinElmer) were performed according to manufacturer’s instructions (Binder et al. 2008 (link)). The fluorescence signal was measured using a PHERAstar FS microplate reader (BMG Labtech, Aylesbury, UK). A minimum of four independently transfected replicates were used for each construct within each experiment, and each experiment was performed on four to five separate occasions with different cell passages. Data were plotted as fold-change responses relative to the response at 0 ng/mL in cells expressing the WT PRLR expression construct. Statistical analyses were performed using two-way ANOVA with Dunnett’s or Tukey’s multiple-comparison tests for pSTAT5 studies and by one-way ANOVA with Sidak’s multiple-comparison tests for pAkt studies