Prostate Cell Culture and Antibody Characterization

BHPrS1 human prostate stromal cells [35 (link)] were cultured in RPMI 1640 (Gibco, Grand Island, NY), 5% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (Gibco). NHPrE1 prostate epithelial cells [36 (link)] were cultured in DMEM/F12 (Gibco) containing either 5% complete fetal bovine serum (FBS) or 5% dextran-coated charcoal-stripped FBS (CS-FBS) (Atlanta Biologicals), 1% penicillin/streptomycin (Gibco), 10ng/ml epidermal growth factor (Sigma Aldrich, St. Louis, MO), 1% insulin-transferrin-selenium (ITS) (Gibco), and 0.4% bovine pituitary extract (Atlanta Biologicals). Primary antibodies against p65, acetyl-p65 (K310) were purchased from Cell Signaling (Beverly, MA), AR (N-20 and C-19) from Santa Cruz Biotechnology (Santa Cruz, CA), AR-V7 from Precision Antibody (Western blotting) (Columbia, MD) and Abcam (IHC)(Cambridge, MA) and, and phospho-p65(S276) from Abcam. Secondary antibodies were purchased from GE Healthcare (Pittsburg, PA).

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Austin D.C., Strand D.W., Love H.L., Franco O.E., Jang A., Grabowska M.M., Miller N.L., Hameed O., Clark P.E., Fowke J.H., Matusik R.J., Jin R.J, & Hayward S.W. (2015). NF-κB and Androgen Receptor Variant Expression Correlate with Human BPH progression. The Prostate, 76(5), 491-511.

Publication 2015

RPMI 1640 penicillin/streptomycin DMEM/F12 epidermal growth factor insulin-transferrin-selenium (ITS) bovine pituitary extract N-20 and C-19)

Antibodies Antibody Biologicals Bovine Charcoal Dextran Epidermal growth factor Epithelial cells Fetal bovine serum Human Insulin Penicillin Prostate Selenium Streptomycin Stromal cells Transferrin

Corresponding Organization : NorthShore University HealthSystem

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Variable analysis

independent variables

Cell culture conditions (RPMI 1640 with 5% FBS and 1% penicillin/streptomycin for BHPrS1 cells, DMEM/F12 with 5% FBS or 5% CS-FBS, 10ng/ml EGF, 1% ITS, and 0.4% bovine pituitary extract for NHPrE1 cells)

dependent variables

Protein expression levels of p65, acetyl-p65 (K310), AR (N-20 and C-19), AR-V7, and phospho-p65(S276)

control variables

Passage number of cell lines (35 for BHPrS1, 36 for NHPrE1)
Cell type (BHPrS1 prostate stromal cells, NHPrE1 prostate epithelial cells)

controls

No positive or negative controls were explicitly mentioned in the protocol.

Annotations

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