Ab74562

For expression in cultured cells, EGFP tag-fused ubiquitin, FLAG-CNPY2, FLAG-MYLIP, MYLIP-His, FLAG-UBE2D1, UBE2D1-His and deletion mutants of FLAG-ARs (full length, 1-920 a.a.; AF-2, 555-920 a.a.; AF-1, 1-673 a.a.) were inserted into the pcDNA3 vector (Invitrogen, Carlsbad, CA, USA). To produce recombinant proteins in E.coli, CNPY2, UBE2D1 and the RING domain of MYLIP (344-445 a.a.) were inserted into pET29a (+) vector (Merck Millipore, DA, Germany) or the pGEX4T1 vector (GE Healthcare, Little Chalfont, England). siRNAs for CNPY2 (CNPY2HSS115810 and 115811) and MYLIP (MYLIPHSS120911 and 120912) were purchased from Invitrogen. Data in this report was shown as results using siCNPY2 (115810) and siMYLIP (120911), which had been more efficient at knockdown or cell growth than siCNPY2 (115811) in 22Rv1 cells. A nonspecific control siRNA pool (siControl) was purchased from Dharmacon (D-001206-13-20). The following antibodies were used: AR (N-20, C-19 or 441, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CNPY2 (ab181215, Abcam, Cambridge, MA, USA), MYLIP (ab74562, Abcam), β-actin (A5441, Sigma, St. Louis, MO, USA), Ub (F-11, Santa Cruz Biotechnology), FLAG (F7425, Sigma), His (D291-3S, MBL, Nagoya, Japan), GST (B-14, Santa Cruz Biotechnology) and GFP (598, MBL).

The total cell lysates were separated by SDS-PAGE gel and followed by Western blot. The images were processed and the integrated optical densities (IOD) of the bands were analyzed by Image Lab 4.0 (Bio-Rad Laboratories, Inc.) software packages. The antibodies used in this experiment were as follows: MYLIP (ab74562, Abcam, USA), E-Cadherin (ab76055, Abcam, USA), ZEB1 (ab181451, Abcam, USA), TWIST (ab49254, Abcam, USA), SNAI1 (13099-1-AP, Proteintech, USA), Vimentin (ab92547, Abcam, USA) and GAPDH (sc-365062, Santa Cruz, USA).