Lightning plus ecl enhanced chemiluminescence substrate

Prostatic organoids and PCa cell lines were lysed in RIPA buffer, supplemented with protease (05892970001; Sigma‐Aldrich) and phosphatase PhoSTOP (PHO SS‐RO; Sigma‐Aldrich) inhibitors. Protein concentrations were determined using Bradford assay (Abcam; ab119216). Equal amounts of proteins were resolved on SDS–PAGE, and transferred onto nitrocellulose membranes using Trans‐blot turbo transfer system (Bio‐Rad).
Membranes were incubated with anti‐CC3 (CST 9664), anti‐cleaved PARP (CST 9544), anti‐HIF1A (CST, 36169), anti‐histone H3 (CST, 4499S), and anti‐ß‐actin (SCBT SC‐47778 or Sigma‐Aldrich A5441) primary antibodies diluted at 1:1,000. Membranes were then incubated with anti‐Mouse IgG (CST 7076S) or anti‐Rabbit IgG (CST 7074S) HRP‐linked antibodies diluted at 1:5,000. Signals were developed using Lightning Plus‐ECL Enhanced Chemiluminescence Substrate (Perkin Elmer; ref: NEL104001EA) and detected using an Amersham™ Imager 600 (GE Healthcare).

Protein lysates were prepared from samples using radioimmunoprecipitation assay buffer, supplemented with protease (05892970001; Sigma-Aldrich) and phosphatase PhoSTOP (PHO SS-RO; Sigma-Aldrich) inhibitors. Protein concentrations in samples were determined using Bradford assay (Abcam, ab119216). Equal amounts of proteins were resolved on SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes using Trans-blot turbo transfer system (Bio-Rad).
The following primary antibodies were used at a 1:1000 dilution: HIF1A (CST 36169), Eno1 (Abcam, ab155102), Hk2 (CST, 2867), Gapdh (CST, 2118), Pten (CST, 9559), SOX2 (CST, 3579), EZH2 (CST, 5246), Ldha (Thermo Fisher Scientific, PA5-27406), phospho-Akt (S473) (CST, 4060S), ß-actin (SCBT, SC-47778), ONECUT2 (Proteintech, 21916-1-AP), histone H3 (CST, 4499S), and vinculin (SCBT, SC-25336). Anti-mouse IgG (CST, 7076S) and anti-rabbit IgG (CST, 7074S) horseradish peroxidase–linked antibodies were used at a dilution of 1:5000. Lightning Plus-ECL Enhanced Chemiluminescence Substrate (Perkin-Elmer, ref. NEL104001EA) was used to develop the signal, which was detected using the Amersham Imager 600 (GE Healthcare).

Whole prostates, FACS-sorted cells, and IMR-90 cells were lysed in ice-cold radioimmunoprecipitation assay buffer supplemented with protease (05892970001; Sigma-Aldrich) and phosphatase PhoSTOP (PHO SS-RO; Sigma-Aldrich) inhibitor cocktails. Protein concentrations of cell lysates were determined by Bradford assay (Abcam; ab119216). Equal amounts of proteins from samples were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Trans-Blot Turbo Transfer System, Bio-Rad). Membranes were then incubated in blocking buffer (5% nonfat dry milk in tris-buffered saline/0.1% Tween 20) for 1 hour at room temperature and then probed with the following antibodies prepared at a dilution of 1:1000: Bcl-xL (CST 2764S), phospho-IKKα (Ser¹⁸⁰)/IKKβ (Ser¹⁸¹) (CST 2681), IκBα (CST 4814), cleaved caspase 3 (CST 9664), β-actin (SCBT; sc-47778), histone H3 (CST; 4499S), and β-tubulin (IGBMC antibody facility, TUB-2A2). Anti-mouse immunoglobulin G (IgG) (CST 7076S) and anti-rabbit IgG (CST 7074S) horseradish peroxidase (HRP)–linked antibodies were used at a dilution of 1:5000. Lightning Plus-ECL, Enhanced Chemiluminescence Substrate (Perkin Elmer; reference: NEL104001EA) was used to visualize proteins on membranes, and images were captured using Amersham Imager 600 (GE Healthcare).