A minimum of two different siRNAs were used for each gene. a Western blot analysis of ARHGDIA, TAGLN2, YWHAZ and AR protein levels in LNCaP cells transfected with siRNAs (1 or 10 nM) targeting (i) YWHAZ, (ii) TAGLN2 or (iii) ARHGDIA for 72 h. 7 AR-modulatory miR target siRNA silencing phenocopies miRs. Images were acquired on a Thermo Scientific myECL Imager (Product # 62236).įig. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075). The membrane was probed with an Androgen Receptor monoclonal antibody (Product # MA5-13426) at a dilution of 1:200 overnight rotating at 4☌, washed in TBST, and probed with an HRP- conjugated goat anti-mouse IgG at a dilution of 1:20,000 for 1 hour. Eluted sample and 20 µg of LNCaP whole cell lysate (loading control) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with StartingBlock T20 (Product # 37543) for 1 hour. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Antigen-antibody complexes were formed by incubating 250 µg of LNCaP whole cell lysate with 2 µg of an Androgen Receptor monoclonal antibody (Product # MA5-13426) overnight on a rocking platform at 4C. Immunoprecipitation of Androgen Receptor was performed on LNCaP cells.
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