Western blot substrate kit

Lysates were prepared by suspending cells in Laemmli sample buffer (Sigma-Aldrich) and heated at 100 °C for 10 min, and resolved by SDS-PAGE (Bio-Rad, Hercules, CA, USA). After proteins were transferred to PVDF membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), primary antibodies were used for immunodetection: rabbit anti-AR (1:1000) [51 (link)] or mouse anti-actin (1:4000, Sigma-Aldrich). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (1:5000, Millipore, Bedford, MA, USA), as well as the Western Blot Substrate kit (Bio-Rad) were used to detect chemiluminescence using a BioRad ChemiDoc^™ XRS+ imaging system.

Lysates were prepared by suspending cells in Laemmli sample buffer (Sigma-Aldrich) and heated to 100 °C for 10 min, and resolved by SDS-PAGE (Bio-Rad, Hercules, CA, USA). Proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and primary antibodies were used for immunodetection: rabbit anti-AR (1:1000) [28 (link)] or mouse anti-actin (1:4000, Sigma-Aldrich) or rabbit anti-p-JNK, JNK, p-ERK, ERK, p-p38, p38 and Bip/GRP78 (1:1000, Cell signaling Technology, Inc., Danvers, MA, USA). Secondary anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase (1:5000, Millipore, Bedford, MA, USA), as well as the Western Blot Substrate kit (Bio-Rad) were used to detect chemiluminescence using a BioRad ChemiDoc™ XRS+ imaging system.

Cells were collected in Laemmli sample buffer (Sigma-Aldrich, St. Louis, MO, USA) and heated to 100 °C for 10 min. Proteins were resolved by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), using a wet blotter (Bio-Rad). Membranes were blocked with 5% non-fat milk and then probed with primary antibodies: rabbit anti- p-ERK, ERK, fibronectin, p-Smad2, Smad2 (1:1000, Cell Signaling Technology, Inc., Danvers, MA, USA) or mouse anti-β-actin (1:4000, Sigma-Aldrich) or rabbit anti-AR (1:1000) [20 (link)] overnight at 4 °C. Membranes were washed and probed with secondary antibodies conjugated to horseradish peroxidase (Millipore, Bedford, MA, USA), and developed with the Western Blot Substrate kit (Bio-Rad) by detecting chemiluminescence using a Bio-Rad ChemiDoc^™ XRS+ imaging system.