Anti ar 441

Tumors were fixed using formalin and paraffin embedded tissue blocks were dewaxed, rehydrated, and endogenous peroxidase activity blocked. Antigen retrieval was performed in sodium citrate buffer (0.01 mol/L, pH 6.0) in a microwave oven at 1,000 W for 3 min and then at 100 W for 20 min. Nonspecific antibody binding was blocked by incubating with 10% fetal bovine serum in PBS for 30 min at room temperature. Slides were then incubated with anti-Ki-67 (NeoMarker), anti-AR (441) (Santa Cruz), and anti-hnRNPA1 (Sigma) at 4°C overnight. Slides were washed and incubated with biotin-conjugated secondary antibodies for 30 min, followed by incubation with avidin DH-biotinylated horseradish peroxidase complex for 30 min (Vectastain ABC Elite Kit, Vector Laboratories). The sections were developed with the diaminobenzidine substrate kit (Vector Laboratories) and counterstained with hematoxylin.

For subcellular protein extracts, cells were fractionated using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermofisher Scientific; Cat#78840), per the manufacturer’s protocol. The proteins were separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Cat# IPVH00010, Fisher Scientific). The membranes were blocked in a solution of PBS, 0.1% Tween 20, and 5% BSA for 1 hour. Following the blocking step, the membranes were incubated overnight at 4°C with one of the following antibodies: anti-AR (441, Santa Cruz Biotechnology; Cat# sc-7305), anti-Histone H3 (Cell Signaling; Cat# 9715), or anti-tubulin (Covance; Cat# MMS-489P). After three washes with PBS, 0.1% Tween 20, the membranes were incubated for 1 hour with either goat anti-mouse horseradish peroxidase-conjugated (HRP) IgG (Cat# 7076S, Cell Signaling) or donkey anti-rabbit HRP-conjugated IgG (Cat# NA9340, Amersham). The membranes were then extensively washed with PBS, 0.1% Tween 20, and the immunoreactive bands were visualized using enhanced chemiluminescence detection reagents (Cat#1705060, Bio-Rad) according to the manufacturer’s instructions.