Journal: Nucleic Acids Research
Article Title: Positive feedback loop mediated by protein phosphatase 1α mobilization of P-TEFb and basal CDK1 drives androgen receptor in prostate cancer
Figure Lengend Snippet: AR stimulates PP1α recruitment and mobilization of P-TEFb. ( A and B ) LNCaP cells in CDS medium were treated for 2 h (A) or 2–8 h (B) with androgen (10 nM DHT) as indicated, followed by ChIP-qPCR for AR and PP1α binding to the indicted AREs. ( C ) LNCaP cells in CDS medium were treated for 4 h with PP1α inhibitor tautomycin (Tau, 200 nM) and DHT, followed by ChIP-qPCR. ( D ) LNCaP in CDS medium were treated overnight with PP2A-specific inhibitor fostriecin (Fos, 100 nM) and DHT, followed by ChIP-qPCR. ( E ) LNCaP cells in CDS medium were treated for 4 h with tautomycin and DHT as indicated, followed by isolation of cytoplasmic, nuclear and chromatin fractions, with HSP90, FOXA1 and H3 used as marker proteins for the cellular fractions, respectively (right panel). The chromatin fraction was then immunoblotted for the indicated proteins (left panel). ( F–H ) LNCaP cells in CDS medium were transfected with control siRNA, AR-specific siRNA or two independent PP1α siRNAs (20 nM) for 3 days, followed by treatment with DHT (10 nM). Total proteins were normalized and immunoblotted (F); cells were harvested for ChIP-qPCR analysis (G); or qRT-PCR analysis was carried out for PSA of KLK2 pre-mRNA (values normalized to the basal activity without DHT, set to 1) (H).
Article Snippet: Antibodies and immunoblotting The sources for the antibodies and control IgGs were as following: AR (Santa Cruz, Cat. sc-816); pAR-S81 (EMD Millipore, Cat. 07–1375); pAR-S308 (Santa Cruz, Cat. sc-26406); pRNA Pol II Ser2 (Abcam, Cat., ab5095); pRNA Pol II Ser5 (Abcam, Cat. ab5131); CDK1 (Cell Signaling, Cat. 9112); pCDK1-T161 (Cell Signaling, Cat. 9114); CDK9 (Santa Cruz, Cat. sc-8338); Cyclin T1 (Santa Cruz, Cat. sc-10750); BRD4 (Bethyl, Cat. A301-985A); p300 (Santa Cruz, Cat. sc-585); Histone 3 (Abcam, Cat. ab1791); H3K27Ac (Abcam, Cat. ab4729); pH3-Ser10 (EMD Millipore, Cat. 06–570); FoxA1 (Abcam, Cat. Ab23738); PSA (Meridian Life Science, Cat. K92110R); Flag-M2 (Sigma-Aldrich, Cat. F3165); HA (Cell Signaling, Cat. 3724); β-Tubulin (EMD Millipore, Cat. MAB3408); β-Actin (Abcam, Cat. Ab6276); GAPDH (Abcam, Cat. Ab9485); Hsp90 (Santa Cruz, Cat. sc-69703), PP1 (Santa Cruz: Cat. sc-443; Cat. sc-6104; and Cat. sc-6105), and control IgGs (Santa Cruz: normal rabbit IgG, Cat. sc-2027; and normal goal IgG, Cat. sc-2028).
Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Isolation, Marker, Transfection, Quantitative RT-PCR, Activity Assay