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Pho ss ro

Manufactured by Merck Group

The PHO SS-RO is a laboratory equipment product manufactured by the Merck Group. It is designed to perform reverse osmosis, a water purification process. The core function of this product is to remove dissolved salts and other impurities from water, producing high-purity water suitable for various applications in research and scientific settings.

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4 protocols using pho ss ro

1

Western Blot Analysis of Nuclear Proteins

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Cells were harvested at indicated time points. RIPA buffer (Thermo Fisher 89901) containing protease and phosphatase inhibitors (Sigma–Aldrich PHOSS-RO, CO-RO) was used to lyse cells. For nuclear fractionation experiments, the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific 78835) were used to separate the nuclear and cytoplasmic cellular fractions. All samples were sonicated and then standardised using a BCA assay. Samples were run in a 4–12% NUPAGE Bis-Tris protein gel at 120 V then transferred to a PVDF membrane, blocked in 5% milk in TBST, and primary antibody (AR: 1:1000, Millipore PG-21; ERα: 1:1000, Cell Signaling 8644; β-actin: 1:50,000, Cell Signaling 12262 S; LaminB1: 1:1000, Cell Signaling 12586; GAPDH: 1:1000, Cell Signaling 2118L) was added overnight. Anti-rabbit secondary antibody (1:10,000, Cell Signaling 7074S) was then added and blots were visualised using ECL Prime on a ChemiDoc Imaging System. ImageJ was used for the quantification of western blots.
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2

Protein Expression Analysis of Prostate Cancer

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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).
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3

Comprehensive Protein Expression Analysis

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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).
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4

Western Blot Analysis of Protein Signaling

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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).
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