Ecl hrp detection reagents

Protein extracts (1 technical replicate per experiment) were prepared in passive lysis buffer (Promega Ltd, Madison, WI). Protein concentrations were estimated at OD595 using the BioRad protein assay (BioRad Laboratories Ltd, Hemel Hempstead, UK). 20 µg protein were separated by SDS-PAGE, blotted onto PVDF filter (Millipore, Watford, UK) and blocked overnight in PBS-T containing 5% non-fat dried milk. Antibodies used were Androgen Receptor (ab9474) and TP53 (ab7757), obtained from Abcam (Cambridge, UK), MXD1 (sc-222), SP3 (sc-644), MYC (sc-764), obtained from Santa Cruz Biotechnology Inc (Heidelberg, Germany) and JUN (9165), obtained from New England Biolabs UK (Hitchin, UK) Primary antibodies were detected with HRP-conjugated secondary. HRP was detected using ECL HRP detection reagents (Amersham Pharmacia, Buckinghamshire, UK). All experiments were performed at least twice (2 biological replicates).

Cytoplasmic and nuclear extracts were prepared with NE-PER extraction reagents (Thermo Fisher Scientific, York, UK). Whole cell extracts were prepared with Dynabeads lysis buffer (Thermo Fisher Scientific, York, UK). Proteins were separated by SDS-PAGE, blotted onto PVDF filter (Millipore, Watford, UK) and blocked overnight in PBS-T containing 5% non-fat dried milk. Antibodies were ab67335, PGK1; ab131591, DJ1; ab4182, TRF2; and ab97433, Ku80 (Abcam, Cambridge, UK). HRP-conjugated secondary was detected using ECL HRP detection reagents (Amersham Pharmacia, Buckinghamshire, UK). Experiments were performed at least twice.