Tris glycine gradient gel

4-20% Tris glycine gradient gels (Biorad) were used for SDS-PAGE followed by Western blot. For immunoprecipitation, cells were lysed in immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, protease inhibitor cocktail (Sigma Aldrich)), mixed with 15 μl protein A/G agarose beads (SantaCruz Biotechnology) and 3 µg anti-polyglutamylation (GT335) antibody, and incubated for 4 h at 4°C subjected to end to end rotation. The beads were washed twice in IP buffer containing 400 mM NaCl. Proteins were eluted by boiling with 2X gel loading dye followed by western blot.

Cells were seeded at a density of 10 000 cells/well in 96-well plates and were incubated in either normal media or CSS media. For pretreatment experiments, cells were treated with KV-49g or INDO for 24 h followed by the addition of ARN or ODM and incubated for a further 72 h. Cell viability was determined by the MTS tetrazolium dye assay as described previously.^{14 (link)} No intrinsic absorbance was noted with ARN, ODM, KV-49g, or INDO in the MTS assay.
Western Blotting. Cells were washed once with PBS and lysed with RIPA buffer (ThermoFisher). Proteins were quantified via BCA assay (ThermoFisher no. PI-23221) and then were resolved by SDS-PAGE on 4–20% Tris-Glycine gradient gels (BioRad). After transfer to nitrocellulose membranes, blocking was conducted for 1 h in Tris-buffered saline (10 mM Tris-HCl, 100 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TBST) and 1% BSA. Samples were probed overnight at 4 °C with anti-aldo-keto reductase 1C3 enzyme (AKR1C3; Sigma; no. A6229, mouse mAb, 1:500), anti-prostate-specific antigen (PSA; Cell Signaling Technology; no. 5877S, rabbit mAb, 1:1000), or anti-β-actin (Sigma; no. A5441, mouse mAb, 1:1000), followed by incubation with anti-mouse (Sigma; no. SAB4600224) or anti-rabbit (Perkin-Elmer; no. NEF812001EA) secondary antibody (1:2000) horseradish peroxidase conjugate for 2 h, and bands were quantified by densitometry using ImageJ software.