Ar441

Cellular protein extracts were resolved on SDS–PAGE and proteins were transferred to nitrocellulose membranes. After blocking for 1 hour at room temperature in 5% milk in PBS/0.1% Tween-20, membranes were incubated overnight at 4°C with the indicated primary antibodies [AR441, (SC-7305, Santa Cruz Biotechnology, Santa Cruz, CA); AR-V7 (AG10008, Precision antibody); Tubulin (T5168, Sigma-Aldrich, St. Louis, MO)]. Tubulin was used as loading control. Following secondary antibody incubation, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Millipore, Billerica, MA) [43 (link)].

Cells plated onto poly-L-lysine coated coverslips were fixed according to pH-shift protocol as described [57 (link)] and stained for AR (AR441, Santa Cruz Biotechnology) and LMTK2 (HPA010657, Sigma-Aldrich). Coverslips were mounted onto a slide using Prolong Gold with DAPI (Invitrogen). The cellular signal was visualized using a PlanApo 60 ×, 1.42 NA oil immersion objective of an Olympus IX71 inverted microscope (Olympus, Center Valley, PA) coupled to a VT-Infinity 3 confocal system (VisiTech International, Sunderland, UK). For each sample, multiple coverslips were imaged (≥30 cells per coverslip) under identical settings.

Cells were grown in 100 mm dishes and induced with Dox (500 ng/ml) for 60 h and treated with DHT (10 nM) and epoxomycin (500 nM) for the last 12 h. Cells were lysed on the dish in tandem Ubiquitin binding entity (TUBE) lysis buffer (20 nM Na₂HPO₄, 20 nM NaH₂PO₄ (pH 7.2), 50 mM NaF, 5 mM tetra-sodium pyrophosphate, 10 nM β-glycerophosphate, 2 mM EDTA, 1 mM DTT, 1% NP-40) supplemented with fresh N-ethyl maleimide (NEM; 10 mM), and protease inhibitors and then 40 μl of GST-TUBE2 (Lifesensors, Inc., Malvern, PA, USA) was added. Cells were incubated on ice for 15 mins and then clarified (12,000 rpm for 10 min). A DC protein assay was performed to determine protein concentration. Glutathione (GST) affinity resin (GE Healthcare, Inc.; 100 μl) was equilibrated according to the provided protocol. GST resin was added to the cell lysate/TUBEs (2.5 mg protein) and incubated at 4°C for 4 h. GST-resin was collected by centrifugation (1000× g, 4°C) for 5 min, unbound sample was removed and resins washed three times with TBST. Product was eluted in 2× Laemmli buffer and loaded on a 10% SDS-PAGE gel for electrophoresis and transferred to 0.45 mM PVDF (Immobilon-P). Western hybridization was performed using the following antibodies: AR(441) (1:500) (Santa Cruz Biotechnology) and Ubiquitin (1:1000) (Dako).