Goat anti mouse igg secondary antibody

Total protein was extracted from PC12 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Following centrifugation for 10 min at 4˚C (12,000 x g), cell supernatant was collected and the protein concentration was estimated using a BCA assay kit (Beyotime Institute of Biotechnology). A total of 20 µg protein per lane was separated by 10% SDS-PAGE gel and transferred onto PVDF membranes. Membranes were blocked with 5% skimmed milk at room temperature for 2 h followed by incubation with primary antibodies at 4^˚C overnight. FABP4 (1:5,000; cat. no. ab92501), cleaved-caspase-3 (1:500; cat. no. ab49822) and caspase-3 (1:2,000; cat. no. ab184787) antibodies were obtained from Abcam, GRP78 (1:1,000; cat. no. sc-13539) and CHOP (1:1,000; cat. no. sc-7351) antibodies were purchased from Santa Cruz Biotechnology, Inc. β-actin (1:5,000; A1978) antibody was obtained from Sigma-Aldrich (Merck KGaA). Horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ab6721; Abcam) and goat anti-mouse IgG secondary antibodies (1:5,000; cat. no. ab6789; Abcam) were used for detection (room temperature; 2 h). The protein bands were visualized with an Enhanced Chemiluminescence Detection kit (Thermo Fisher Scientific, Inc.). Protein expression levels were semi-quantified using Image-Pro Plus software version 6.0 (Roper Technologies, Inc.).

SiHa cells were lysed in cell RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA). Bradford Protein Assay Kit (Beyotime) was used to measure the protein concentration. Then, an equal amount of protein was separated using 10% SDS polyacrylamide gel and transported onto polyvinylidene fluoride membranes (PVDF, Thermo) in 2 h. The PVDF membranes were blocked in 5% non-fat milk in TBST at room temperature for 1 h. Later on, the membranes were washed in TBST 3 times and incubated with primary antibodies overnight at 4 °C: anti-Bax (Abcam; ab32503) (1:1000), anti-Bcl-2 (Abcam; ab32124) (1:1000), anti-active caspase 3 (Abcam; ab2302) (1:1000), anti-c-Kit (Abcam; ab216450) (1:1000), anti-c-myc (Abcam; ab32072) (1:1000), anti-cyclin D1 (Abcam; ab16663) (1:1000), anti-β-actin (Abcam; ab8227) (1:1000), After washing with TBST for three times, the membrane was incubated with secondary antibody goat anti-mouse IgG secondary antibody (Abcam; ab6789) (1:5000), goat anti-rabbit IgG secondary antibody (Abcam; ab205718) (1:5000) before determined by chemiluminescence. Finally, the PVDF membranes were incubated with ECL reagent (Santa Cruz Biotechnology) to detect the blots. The density of blots for proteins were normalized to β-actin.

Cells treated with culture medium containing 1, 10, and 100 μmol/L mTOR agonist MHY1485 (MCE) were cultured continuously for 96 h; control cells were incubated in normal culture medium. The cells were then maintained at 37°C in 5% CO₂ and 95% air. Cells were harvested 96 h after treatment, and total RNA was isolated using TRIzol reagent (Invitrogen). The RNAs were reverse transcribed by SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). The sequences of primers for qPCR are listed in Table 1. Total protein from chicken granulosa cells was extracted with the Total Extraction kit (Sigma). The procedures described for Western blotting were repeated using primary antibodies against mTOR (Abcam, 1:2,000 dilution), P-mTOR (Abcam, 1:1,500 dilution), S6K (Abcam, 1:1,000 dilution), and p-S6K (Abcam, 1:1,000 dilution) and goat anti-mouse IgG secondary antibody (Abcam, 1:5,000 dilution). An antibody against β-actin (Abcam, 1:1,000 dilution) was used as an internal control. Glyko Bandscan 5.0 (Beijing Sage Creation Science Co., Ltd., Beijing, China) image analysis software was used for densitometric analysis.