Cyclescript rt premix

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, #15596026) according to the manufacturer's protocol. cDNA was prepared to employ a CycleScript RT premix (Bioneer, #K-2044-CFG). All primer sequences are listed in Table 1. Expression levels were calculated using a SensiFAST SYBR Lo-ROX Kit (Bioline, #BIO-94020) and a commercial detection system (BioRad, #CFX96).
Total proteins were extracted into RIPA buffer (Thermo Fisher Scientific, #89900) containing a phosphatase inhibitor (Sigma-Aldrich, #4906845001) and a protease inhibitor (Roche, #43693159001), subjected to 10–16% (w/v) Tris-glycine SDS-PAGE, transferred to PVDF membranes using Iblot 2 NC ministacks (Invitrogen, #IB23002), and the membranes blocked with 5% (w/v) skim milk. The primary antibodies were anti-pAMPK (Cell Signaling Technology, #2531S), anti-AMPK (Cell Signaling Technology, #5831S), anti-pACC (Cell Signaling Technology, #3661S), anti-ACC (Cell Signaling Technology, #3662S), and anti-GAPDH (Cell Signaling Technology, #2118S) diluted 1:1000 (primary antibodies) or 1:5000 (secondary antibodies) in TBST (Biosesang, #HT2007) containing 5% (w/v) skim milk. The membranes were then incubated with a peroxidase-conjugated anti-rabbit secondary antibody and signals quantitated using the Immobilion Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500).

Total RNA was isolated from wild-type and CRBN−/− MEFs by TRIzol reagent (Invitrogen), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized using CycleScript RT PreMix (Bioneer). mRNA levels were measured using TB Green™ Premix Ex Taq™ (TaKaRa) and Thermal Cycler Dice Real-Time System. The following primers were used for amplification. p21, forward: 5′ AAT CCT GGT GAT GTC CGA CC -3′, and reverse: 5′-AAA GTT CCA CCG TTC TCG G-3′; 18s rRNA, forward: 5′-GTA ACC CGT TGA ACC CCA TT-3′, and reverse: 5′-CCA TCC AAT CGG TAG TAG CG-3′. Expression was normalized to 18 s rRNA levels.

Ovaries were punctured with a sterile blade and germinal vesicle (GV) oocytes were collected in M2 medium supplemented with 2.5 μM milrinone (Sigma-Aldrich, Missouri, USA) to maintain them at the germinal vesicle (GV) stage. After PMSG injection, we used a syringe to penetrate the ovary to obtain granulosa cells. Granulosa cells were washed with M2 medium and transferred to PCR tubes. Total RNA from the mouse ovaries were prepared using a miRNeasy Mini Kit (Qiagen, Hilden, Germany). Briefly, ovaries were ground in liquid nitrogen and homogenized in RLT buffer (Qiagen, Hilden, Germany) supplemented with 1% β-mercaptoethanol. The supernatant, mixed with 70% ethanol, was transferred into mini spin columns and total RNA was collected. For the isolation of total RNAs from GV oocytes and cumulus cells, ~100 GV oocytes and cumulus cells from three mice were prepared and the total RNA was isolated using a miRNeasy Micro Kit (Qiagen, Hilden, Germany), in the same manner as the miRNeasy Mini Kit. First-strand cDNA was synthesized with CycleScript RT PreMix (Bioneer, Seoul, Korea). Gapdh was used as an internal standard. The primers for the amplification of CCP1 were described in Kim et al.[8 (link)] and Ccp4 (also known as Agbl1) and CCP6 (also known as Agbl4) were described by Kalinina et al. [10 (link)].PCR products were run on 1.5% agarose gels and visualized using UV illumination.