Ubiquitin

Immunoblotting and co-immunoprecipitation analyses were performed as previously described^{21 (link)}. Briefly, immunoblotting samples were prepared with RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate and protease and phosphatase inhibitors at pH 7.5). Co-immunoprecipitation samples were prepared with IP lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 5% glycerol, 1 mM EDTA with protease and phosphatase inhibitors). Then, 500 μg of protein was incubated with each antibody for 24 h at 4°C, followed by incubation with protein A-sepharose or protein G-agarose beads (Roche Applied Science) for 2 h. Samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Antibodies against phospho-IRS-1(S302), phospho-IRS-1(S612), Akt, ERK, phospho-Akt, and phospho-ERK were purchased from Cell Signaling Technology, and antibodies against Flag, HA, ubiquitin, GAPDH, and phospho-IRS-1(Y628) were purchased from Santa Cruz Biotechnology (CA, USA). The IRS-1 antibody was purchased from BD Transduction Laboratories (CA, USA), and the phospho-IRS-1(Y608) antibody was purchased from Merck Millipore (MA, USA).

The cells were lysed with PRO-PREP Protein Extraction Solution (iNtRON Biotechnology Inc., Korea) and the following antibodies were used for western blot analysis: DBC1 (Bethyl Laboratories, Mongomery, TX), AR (Santa Cruz Biotechnology, Santa Cruz, CA), P53 (Novocastra, Newcastle, UK), acetylated-P53 (Cell Signaling Technology, Beverly, MA), P21 (Santa Cruz Biotechnology, Santa Cruz, CA), P27 (Santa Cruz Biotechnology, Santa Cruz, CA), BAX (Santa Cruz Biotechnology, Santa Cruz, CA), BCL-2 (Santa Cruz Biotechnology, Santa Cruz, CA), TGFβ (Cell Signaling Technology, Beverly, MA), NFκB (Santa Cruz Biotechnology, Santa Cruz, CA), PCNA (Santa Cruz Biotechnology, Santa Cruz, CA), Rho A (Santa Cruz Biotechnology, Santa Cruz, CA), Ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA), PARP1 (Santa Cruz Biotechnology, Santa Cruz, CA), and actin (Sigma, St. Louis, MO). Western blot images quantified with ImageJ software. For IP, polyclonal anti-DBC1 or anti-AR antibodies were cross-linked to Dynabeads-protein A (Invitrogen, Carlsbad, CA). DBC1 or AR antibodies were immobilized with Dynabeads-protein A for 20 min and then incubated with cell lysates for 1 h at 4 °C. DBC1- or AR-IP complexes were washed three times and the proteins were eluted. Thereafter, we performed western blotting.

Animal care and handling were performed according to the guidelines set by the World
Health Organization (Geneva, Switzerland). Eight-day-old (P8) female CD-1 mice were
purchased from Charles River Laboratories. Ovaries were harvested, transferred in
sterile flat-bottom 96-well plates with 100 µl MEM (+ L-Glu, Gibco) supplemented with
5% FBS, 0,4% BSA (w/v), Pen/Strep and 70 µM Br-cAMP and cultured in an incubator at
37°C with 5% CO₂.
Ovaries were treated overnight with either DMSO or CHX (50 µg/mL) prior following
experiments. IRR ovaries were exposed to 1.5 Gy of γ-irradiation on a rotating
turntable in a ¹³⁷Cs irradiator, at a dose rate of 2.387 Gy/min. For
inhibition of Chk2 in ovary culture the inhibitor BLM-277 (Merck Millipore, 220486)
was used 2 hr prior γ-irradiation in indicated concentrations.
The following antibodies were used for detection of endogenous protein of ovary
samples by Western Blotting: Msy2 (Santa Cruz, N-13), Ubiquitin (Santa Cruz, P4D1),
p63 (Santa Cruz, H-129) and β-Actin (Santa Cruz, C4).