Western Blot Analysis of PSMA Expression

Cell pellets were lysed on ice in Mammalian Protein Extraction Reagent (MPER, Thermo Fisher, 78,503) containing Halt Protease Inhibitor Cocktail (Thermo Fisher, 87,786). Lysates were quantified using the Pierce BCA Protein Assay kit (Thermo Fisher 23,225). 10 μg of LNCaP and 50 μg other PCA cell line samples were then denatured (95 °C for 5 minutes), separated by SDS-PAGE (4–20%), and transferred to a nitrocellulose membrane. Membranes were blocked in 5% non-fat milk, dissolved in tris-buffered saline-tween 20 (TBST), for 1 hour at room temperature prior to primary antibody incubation. PSMA protein expression was evaluated using a mouse monoclonal, anti-human PSMA antibody (Novus Biologicals, NBP1–45057) with demonstrated reactivity to canine PSMA [23 (link)]. The PSMA antibody was diluted to 1 μg/mL and membranes were incubated overnight at 4 °C, followed by secondary labelling using species a specific HRP-linked antibody (Cell Signaling) for 1 hour at room temperature. Membranes were then developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher 34,094) and ChemiDoc XRS+ molecular imager system. All membranes were re-probed for β-actin (Abcam, ab6276) for use as loading control.