Bicinchoninic acid protein assay kit

Cells and tissues were lysed by RIPA buffer (Beyotime Biotechnology, Beijing, China) with protease inhibitor cocktail (B14002, Biotool), and total protein concentration was measured using a bicinchoninic acid protein assay kit (Tiangen Biotech, Beijing, China). Western blotting was performed with primary antibodies against β-actin (catalog no. ab6276; 1:1000; Abcam), phosphorylated p70S6K (Thr389) (catalog no. 9234; 1:1000; CST), p70S6K (catalog no. 2708; 1:1000; CST), phosphorylated 4EBP1 (Thr70; catalog no. 2983; 1:1000; CST), 4EBP1 (catalog no. 9644; 1:1000; CST), phosphorylated Akt (S473;catalog, 4060; 1:1000; CST), Akt (catalog no. 4685; 1:1000; CST), phosphorylated mTOR (catalog no. 2971; 1:1000; CST), mTOR(catalog no. 2983; 1:1000; CST), phosphorylated ERK (catalog no. 4370; 1:1000; CST), ERK (catalog no. 4695; 1:1000; CST), PCNA (catalog no. 13110; 1:1000; CST), vimentin (catalog no. 5741; 1:1000; CST), E-cadherin (catalog no. sc-8426; 1:500; Santa Cruz) and N-cadherin (catalog no. sc-8424; 1:500; Santa Cruz). Western blots shown in the accompanying figures are derived from three independent experiments.

The total protein concentration derived from NSCLC cells was tested using a bicinchoninic acid protein assay kit (Tian Gen). In brief, 30 μg total protein was added to the relevant sample wells, electrophoretically separated using a 10% sodium dodecyl sulfate‐polyacrylamide gel, and transferred to a polyvinylidene fluoride membrane (Bio‐Rad, USA) through a semi‐dry electrophoretic transfer. The membrane was immersed in the 5% melted skim milk powder, and then the membranes were probed with the primary antibodies on a shaker at 4°C overnight. The next day, the membranes were washed three times with phosphate‐buffered saline containing Tween‐20 and incubated with the corresponding secondary antibodies for 2 h at room temperature. Two minutes after covering the film with a luminescent developer, a gel‐imaging system was used to visualize the protein bands.

Total protein was extracted from the uterus and the PECs using radioimmunoprecipitation assay lysis buffer supplemented with phenylmethanesulfonyl fluoride (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions and detected by the Bicinchoninic Acid Protein Assay Kit (Tiangen Biotech Co., Ltd., Beijing, China). Approximately 50 μg of protein was separated by electrophoresis on polyacrylamide gels and subsequently transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk powder for 1.5 h and incubated overnight at 4 °C with the following primary antibodies: anti-ER-α (1:1000; ab3575, Abcam, Shanghai, China), anti- ER-β (1:1000; ab3576, Abcam), anti-PR (1:200; sc-539, Santa Cruz Biotechnology, Shanghai, China), anti-GAPDH (1:5000; Abcam) and anti-actin (1:5000; Abcam). After incubation, the membranes were washed three times with TBST and incubated with anti-rabbit/mouse IgG antibody (1:2000; CWBIO, Beijing, China) for 2 h at 37 °C, followed by washing with TBST. Finally, the membranes were immersed in a high-sensitivity luminescence reagent (BeyoECL Plus; Beyotime), exposed to film using a FusionCapt Advance FX7 (Beijing Oriental Science and Technology Development Co., Ltd., Beijing, China) and analyzed using Ipp 6.0 (Image Pro-Plus 6.0; Media Cybernetics).

Protein isolation was done using radioimmunoprecipitation assay buffer (CWBio, Beijing, China), and protein concentration was detected using a bicinchoninic acid protein assay kit (Tiangen, Beijing, China). Then, an equal amount of proteins was split by sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) electrophoresis and blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked using 5% nonfat milk for 1 h at indoor temperature. Next, the membranes were immunoblotted with primary antibodies against GAPDH (ab181602; Abcam), total p65 (t-p65; ab16502; Abcam), phosphorylated p65 (p-p65; ab86299; Abcam), B-cell lymphoma-2 (Bcl-2, ab196495; Abcam), BCL2-associated X (Bax, ab180733; Abcam), cleaved-caspase 3 (C-caspase 3, ab49822; Abcam), or total-caspase 3 (t-caspase 3, ab90437; Abcam) overnight at 4°C and then incubated with corresponding secondary antibody (ab6789; Abcam) for 1.5 h at indoor temperature. Finally, the protein bands were exposed through an enhanced chemiluminescence reagent (Vazyme) and analyzed by software Image J.

Epididymal adipose tissues were homogenized in RIPA buffer (Beyotime, Haimen, Jiangsu, China) containing 1% protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA) and 1% phosphatase inhibitor (Sigma-Aldrich, St. Louis, MO, USA) to extract protein. Samples were centrifuged at 12,000× g at 4 °C for 15 min to collect the supernatant. Protein concentration was determined using the bicinchoninic acid protein assay kit (Tiangen Biotech, Beijing, China). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) with a wet transfer apparatus (Bio-Rad, Hercules, CA, USA). After blocking the PVDF membranes with 5% fat-free milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C. Next, the PVDF membranes were washed three times and incubated with a horseradish peroxidase conjugated secondary antibody for 1 h at room temperature. The enhanced chemiluminescence reagent (Millipore, Bedford, MA, USA) and autoradiographic film were used to detect the protein bands. The immunoblotted band intensity was quantified by ImageJ software (version, National Institutes of Health, Bethesda, MD, USA).

After being treated with appropriate doses of each drug for indicated time, cell samples, including GH3, MMQ, and primary pituitary tumor cells, were harvested in sterile Eppendorf tubes and washed with cold PBS for two times. Then, these samples were lysed in lysis buffer (50 mM Tris [pH 7.5], 120 mM NaCl, and 0.5% NP-40) containing protease and phosphatase inhibitors and placed on ice for 30 minutes. After that, they were centrifuged at 12,000 × g for 10 minutes. The total protein concentration of the samples was measured using the bicinchoninic acid protein assay kit (Tiangen Biotech, PA115). The protein samples were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The LAS4000 system was used for imaging, and protein band intensity was quantified by densitometry using the ImageJ software. The following antibodies were used in this study: Tubulin antibody (11224-1-AP; Proteintech Group), S6K1 (Cat#2708, Cell Signaling Technology), p-S6K1-Thr389 (Cat#9234, Cell Signaling Technology), 4EBP1 (Cat#9644, Cell Signaling Technology), p-4EBP1 (Cat#2855, Cell Signaling Technology), Antirabbit IgG, HRP-linked antibody and Anti-mouse IgG, and HRP-linked antibody (#7076; #7074, Cell Signaling Technology).