Mouse anti myc

To perform co-immunoprecipitation, transfected S2 cells were harvested and lysed on ice with lysis buffer (50 mM Tris-HCl/pH 7.4, 150 mM NaCl, 2 mM sodium vanadate, 10 mM sodium fluoride, 1% Triton X-100, 10% glycerol, 10 mM imidazole and 0.5 mM phenylmethylsulfonyl fluoride). Lysates were centrifuged for 15 min at 20,000×g, 4°C and the resulting supernatant was incubated with Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, Paso Robles, California) conjugated to mouse monoclonal anti-GFP clone 20 (Sigma-Aldrich, St. Louis, Missouri) for 4 hr at 4°C. After washing once with lysis buffer, twice with lysis buffer containing 0.1% deoxycholate, and 3 times with lysis buffer lacking Triton X-100, the immunoprecipitates and total lysates were resolved on 7.5% SDS-PAGE gels followed by western blot analysis as previously described (Kim et al., 2013 (link)).
Primary antibodies used in western blotting were mouse monoclonal anti-tubulin (Sigma), mouse anti-Myc (Sigma-Aldrich), mouse monoclonal anti-Aequorea Victoria GFP JL-8 (Clontech), and rabbit anti-phospho-Tyr412-c-Abl (Cell Signaling, Beverly, Massachusetts).

Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Triton-X-100, 2 mM EDTA, 0.01% SDS, 50 mM NaF, 0.1 mM Na₃VO₄, 1×Protease Inhibitor/no EDTA), sonicated (20% amp for 15 s) and centrifuged at 15000 rpm for 15 min at 4 °C. 10 µg of cell lysate was denatured and resolved by 6% homemade SDS-PAGE protein gel [49 (link)]. Proteins were transferred onto nitrocellulose membranes and blocked with 10% skim milk 1× Tris-buffered saline, 0.1% Tween 20 (TBST). The membrane was probed with primary antibody; rabbit anti-AR (Santa Cruz, cat #sc-816), mouse anti-ER $\mathrm{\alpha }$ (Santa Cruz, cat #sc-8002), rabbit anti-PGR (Leica Biosystems, # NCL-L-PGR AB), rabbit anti-FOXA1 (Abcam, cat # ab23738), mouse anti-Myc (Sigma Aldrich, cat #M4439), mouse anti-V5 (Thermo Fisher, cat #46-0705), mouse anti-FLAG (Sigma Aldrich, cat #F3165) or rabbit anti-β-Tubulin (Abcam, cat #ab6046) in 1× TBST/2% skim milk overnight at 4°C. The next day, membranes were probed with anti-mouse immunoglobulins/HRP (Dako, cat #P0447) or goat anti-rabbit immunoglobulin/HRP (Dako, cat #P0448) in 1× TBST/2% skim milk, washed with 1× TBST and detected by Clarity Western enhanced chemiluminescence (ECL) substrate (Bio-Rad) using BioRad ChemiDoc MP Imaging System with Imagining Lab 5.0.

Primary antibodies used in this study were as follows: mouse anti-USP4 (66822; Proteintech1:1000 dilution for WB, 1:300 dilution for IHC); mouse anti-BRCA1 (sc-6954; Santa Cruz; 1:300 dilution for WB); rabbit anti-BRCA1(ab16780; Abcam;1:400 dilution for IHC); rabbit anti-BRCA1 (ab191042; Abcam;1:1000 dilution for WB); rabbit anti-BARD1 (NB100; Novus Biologicals; 1:1000 dilution for WB); mouse anti-BARD1 (sc-74559; Santa Cruz; 1:500 dilution for WB); mouse anti-Flag (F3165; Sigma; 1:5000 dilution for WB); rabbit anti-HA (3924 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-Myc (M4439; Sigma;1:5000 dilution for WB); mouse anti-ubiquitin (3936 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-His (2366 S; Cell Signaling Technology; 1:1000 dilution for WB); mouse anti-α-Tubulin (3873 S; Cell Signaling Technology; 1:3000 dilution for WB), rabbit anti-β-actin (AC026; ABclonal; 1:1000 dilution for WB); rabbit anti-GAPDH(5174 S; Cell Signaling Technology; 1:5000 dilution for WB). For WB, western blots were detected and analyzed using ChemiDoc Imaging system (Bio-Rad). The original WB blots were included in the supplementary information.

Western blot analysis and immunostaining were performed as previously described [40 (link)]. Primary antibodies used in western blot and immunostaining were as follows: mouse anti-Myc (1:5 000, Sigma), mouse anti-Flag (1:5 000, Sigma), mouse anti-V5 (1:5 000, Invitrogen), mouse anti-HA (1:5 000, Sigma), mouse anti-phospho tyrosine (1:1  000, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Ack (1:500), rat anti-Ci (1:500, Developmental Studies Hybridoma Bank, DSHB), rabbit anti-Ex (a gift from Allen Lauhgon, University of Wisconsin), guinea pig anti-Mer (a gift from Richard G. Fehon, University of Chicago) and rabbit anti-lacZ (1:1 000, Invitrogen). Rabbit anti-Ack (556-1073AA) was generated by Abclonal (Wuhan, China). Rabbit anti-phospho Ex (Y679 and Y766) was generated by Abclonal, with peptide as antigen (Y679, ESEKSSHpYGMFQPQK; Y766, SLHSDCDpYVTLPLGD). Rabbit anti-phospho Yki (S168) was generated by Abgent (Suzhou, China) with antigen peptide HHSRARpSSPAC.
For Luciferase reporter assay, the 3xSd2-Luc reporter was described previously [40 (link)]. The Dual-Glo™ luciferase assay system (Promega, Madison, WI, USA) was used according to the manufacturer's instructions.

Cells were grown on a cover-glass, were washed twice with PBS 1X and then fixed with 4% paraformaldehyde in 1 × PBS and permeabilized with 0.2% Triton X-100 in 1 × PBS. After a blocking step for 1 h in 5% BSA, diluted in 1 × PBS-0.05% Tween-20, cells were incubated with the primary antibody mouse anti-Myc (Sigma-Aldrich), diluted 1∶10000 in blocking solution, overnight at 4 °C, and then incubated with a secondary antibody Alexa Fluor^®488 goat anti-mouse IgG (Life Technologies), diluted 1∶1000 in blocking solution, for 1 h at the room temperature. Cells were then analysed with a Leica TCS SP5 confocal microscopy, with LAS lite 170 image software.