β catenin e 5

Three million pCMV Vector or miR-200c transfected SKOV3 cells were collected and lysed in RIPA lysis buffer. Thirty micrograms of protein of each sample was loaded on 10% polyacrylamide gels and run for 1.5 h at 120 V. The following antibodies were used: PD-L1 (E1L3N) (Cell Signaling Technology, Danvers, MA, USA; Cat. n.13684), β-catenin (E-5) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany; Cat. n.sc-7963), and c-Myc (D84C12) (Cell signaling; Cat. n.5605). β-actin (C4) (Santa Cruz; Cat. n.sc-47778) and Lamin B1 (C-20) (Santa Cruz; Cat. n.sc-6216) were used to ensure equal protein loading. HRP conjugated anti-rabbit (SIGMA; Cat. n.A 6154) and anti-mouse (ADVANSTA; Cat. n.R-05071-500) secondary antibodies were used, (1:5000 in 2% BSA) for 30 min. The chemiluminescent signal was detected using WesternBright^® ECL (ADVANSTA, San Jose, CA, USA; Cat. n.K-12045-D20). Densitometry analysis was performed with ImageJ Software (v. 10.2). Immunoblots were repeated three times with the same lysates and with protein lysates derived from three different treatments of olaparib and irradiation. Additional details of immunoblotting conditions can be found in Supplementary Materials and Methods.

The antibodies against HCF1 (sc-390950), HCF2 (sc-393250), Cyclin T1 (H-245), TRAF2 (F-2) and β-Catenin (E-5) were purchased from Santa Cruz Biotechnology. The anti-AFF4 (ab57077) antibody was purchased from Abcam. The anti-AFF1 (A302-344A), -ENL (A302-268A), -AF9 (A300-595A), -ELL2 (A302-505A) and -ELL1 (A301-645A) antibodies were purchased from Bethyl Laboratories. The monoclonal antibodies against HA (3F10) and Flag (M2) were from Roche and Sigma-Aldrich, respectively. The antibodies against CDK9, LARP7 and Brd4 were generated in our own laboratory and have been described previously (14 (link),15 (link)).
The HCF1 cDNA (pCGN-HCF-1; plasmid #53309) with an HA-Tag at the N-terminus and a Myc tag at the C-terminus was purchased from Addgene. The HCF2 cDNA (BC033799) was purchased from transOMIC technologies. The HCF2 cDNA was cloned into expression vectors N-Flag-PRK5M and N-HA-PRK5M, respectively.

The immunohistochemical study was carried out to examine altered protein expression in 180 paraffin-embedded breast tissues as described in previous publications [28 (link)]. All the markers were incubated with the sections overnight at 4°C; the markers included TRPS1 (sc-26974, diluted 1:200; Santa Cruz Biotechnology, CA, USA), P53 (Zhongshan Golden Bridge Biotechnology, ZSGB-Bio, Beijing, China), E-Cadherin (24E10, diluted 1:400, Cell Signalling Technology, USA), mouse monoclonal antibody β-catenin (E-5, diluted 1:500, Santa Cruz Biotechnology, CA, USA), Vimentin ( D21H3, diluted 1:100, Cell Signalling Technology, USA), slug (C19G7, diluted 1:50, Cell Signalling Technology, USA); the second antibody was from IHC reagent kit (Zhongshan Biotechnology Company, Beijing, China). After diaminobenzidine (DAB) staining, the sections were counterstained with hematoxylin. For negative controls, the antibodies were replaced with phosphate buffered saline (PBS).