Enhanced chemiluminescence detection kit

Total cell lysates were collected by RIPA buffer containing commercial protease inhibitors. 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat milk at room temperature for 1 h, followed by incubation with primary antibody prepared in 1 × PBST (PBS with 0.25% Tween 20) (COUP-TFII, 1:1000, PP-H7147-00, R&D systems; CENPF, 1:2000, Ab5, Abcam; FOXM1, 1:2000, #5436, Cell Signaling Technology; E-cadherin, 1:2000, #3195, Cell Signaling Technology; Vimentin, 1:2000, #5741 Cell Signaling Technology; GR, 1:2000, #12041, Cell Signaling Technology and AR, 1:2000, sc-816, Santa Cruz Biotechnology Inc.) at 4 °C overnight. Signals were developed using an enhanced chemiluminescence detection kit (PerkinElmer). For IHC staining, AR (1:1,000) antibody was purchased from Santa Cruz Biotechnology Inc (sc-816) and GFP (1:1,000) antibody was purchased from ThermoFisher (A11122). Uncropped scans of blots are shown in Supplementary Figs 15–17.

Total cell extracts were prepared by incubating cells in lysis buffer (Beyontime, Sanghai, China) containing Protease Inhibitor Cocktail Set III (Merck, Darmstadt, Germany) and PhosSTOP EASYpack (Roche, Basel, Switzerland). The protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were incubated with primary antibodies against VP2, Flag, GRP78, PERK, IRE1, p-IRE1, eIF2α, p-eIF2α, ATF6, CHOP, cleaved caspase-3 and β-actin. HRP-conjugated goat anti-rabbit/mouse IgG was used as secondary antibodies. Immunoreactive bands were visualized utilizing the Enhanced Chemiluminescence Detection Kit (PerkinElmer, Waltham, MA, USA).

Cells were plated at 6×10⁵ cells/well in six-well plates and transfected as described above.
The proteins were extracted, and the total protein concentration was determined by Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA) following manufacturer’s manual. The absorbance was measured by a spectrophotometer (CT-5600; ChromTech, Inc, Apple Valley, MN, USA). Proteins were separated in 8%–13.5% polyacrylamide-SDS gel and transferred onto a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% skim milk in TBST (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween 20), the membranes were incubated with specific antibodies against Calnexin, HuR, HA, c-Myc, caspase-3, -9 (Cell Signaling Technology Inc., Beverly, MA, USA), galectin-3, P-gp, MRP1, Bcl-2 (GeneTex Inc., Hsinchu, Taiwan), and β-actin (Millipore, Billerica, MA, USA) overnight at 4°C. These membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Inc., PA, USA) for 1 h at room temperature. The detection of the signal was performed by incubating blotted membranes with enhanced chemiluminescence detection kit (PerkinElmer Life Sciences, Waltham, MA, USA).

Following administration with different stimulants, total protein was extracted from cells using radioimmunoprecipitation assay lysis buffer in the presence of PMSF (Beyotime Institute of Biotechnology) and protein concentrations were measured using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Protein samples were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bellerica, MA, USA). Following blocking with 5% non-fat milk or 5% bovine serum albumin, membranes were then incubated with primary antibodies against phosphorylated p38MAPK (rabbit monoclonal anti-phospho-p38MAPK; 1:1,000; Cell Signaling Technologies) and total p38MAPK (rabbit monoclonal anti-p38MAPK; 1:1,000; Cell Signaling Technologies), overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit IgG secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h. Western blots were developed using an enhanced chemiluminescence detection kit (PerkinElmer, Inc., Waltham, MA, USA) and quantified by densitometry using Gel-Pro Analyzer software 4.0 (Media Cybernetics, Inc.).

For western blotting, 10–50 μg of protein extracts were separated using 10–12% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose (PVDF) membranes, then blocked overnight with 1 × tris buffered saline buffer containing 5% (w/v) skim milk. Membranes were next incubated with optimal concentrations of primary antibodies: anti-GATA-4 mAb (Abcam), and anti-β actin mAb (Sigma) all diluted in blocking buffer. Membranes were washed and then incubated with appropriate secondary antibodies (goat anti-mouse mAb conjugated with HRP), and visualized using the enhanced chemiluminescence detection kit (Perkin Elmer, MA, USA). The antibodies for GATA-4 (1:100) and Bcl-X_L (1:100) were purchased from Santa Cruz (CA, USA) while Bcl-2 (1:1000) was purchased from Cell Signaling (CA, USA).

Nuclear extract proteins were separated on 10% SDS-polyacrylamide gels and subsequently transferred to PVF membranes. The membranes were blocked for 1 h in 5% BSA in Tris-buffered saline containing 0.01% Tween 20 (TBST). Immunoblotting was performed overnight at 4°C with primary antibodies, anti-ERK1/2, and anti-phospho-ERK1/2 (Cell Signalling) at a dilution of 1 : 2000 and 1 : 1000, respectively. Membranes were incubated with a secondary horseradish peroxidase-conjugated antibody at 1 : 10000 dilutions for 1 h at room temperature. To visualize immunoreactive proteins, the enhanced chemiluminescence detection kit (PerkinElmer, USA) was used. Hypoxanthine-guanine phosphoribosyl transferase (HPRT) (Santa Cruz Biotechnology) was used as the housekeeping protein.

Parts of the tumor specimen were extracted in a homogenization buffer containing 1% NP-40, 0.5% sodium deoxycholic acid, and 0.1% SDS, supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) [24 (link)]. The total protein concentrations of lysates were measured by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using a series of bovine serum albumin as standards. A total of 50 μg per sample was separated on a homemade 10 or 12% SDS-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes and incubated with different primary antibodies (Table S1) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated rabbit (#7074, Cell signaling, Danvers, MA, USA), goat (sc-2354, Santa Cruz, Dallas, TX, USA), and anti-mouse secondary antibodies for one hour at room temperature. The blots were then visualized using an enhanced chemiluminescence detection kit (PerkinElmer Life Sciences Inc. Waltham, MA, USA). Anti-β-actin monoclonal was used as a loading control. Finally, signal intensities were measured using the Luminescent image system (FUJIFILM, LAS-4000, Tokyo, Japan) and Multi Gauge 3.0 software (Fuji, Japan). Densitometry analysis of each bot was normalized by β-actin (n = 5 mice were analyzed in each group).