Protein phosphatase inhibitor

Immunodeficient CB17SC-F scid^−/− female mice (Taconic Farms, Germantown, NY, USA)^{36 (link)} were used to propagate subcutaneously implanted tumors. Tumors were excised, rapidly frozen in liquid nitrogen and pulverized under liquid nitrogen. Total proteins were extracted with cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with protease inhibitors and protein phosphatase inhibitors (Roche, Indianapolis, IN, USA). Thirty-five micrograms of total proteins were used in immunoblotting experiments to detect Sgt1 and GAPDH.
The tumorigenic potential of MEF cells was evaluated by injecting 10⁶ cells (volume, 0.1 ml) into the left flank of immunodeficient CB17SC-F scid^−/− female mice. Tumor volume (cm³) was measured with calipers and determined as previously described.^{37 (link)} Tumor volume was measured biweekly at the initial observation of the tumor growth until endpoint criteria were achieved. Tumor volume was calculated according to the following formula: (π/6) · d³, where d represents the mean diameter.^{37 (link)} Mice were humanely killed at the endpoint. The endpoint criteria met by this study were follows: a tumor volume of 2.24 cm³, ulceration of the tumor and dehydration or poor condition of the animal.

Approximately 20 mg of cerebral cortex tissues was carefully dissected and lysed in 10 volumes (wt/vol) of RIPA buffer containing protease inhibitors or a protein phosphatase inhibitor (Roche, Basel, Switzerland). After centrifugation at 13,000 × g for 10 min at 4°C, the supernatant was preserved. Protein concentrations were determined by BCA assay. The protein concentration of each sample was the same as the lysate buffer. The samples were loaded into the SDS-PAGE and transferred onto nitrocellulose membranes (295 mA, 1.5 h). After incubation with 5% skim milk powder at room temperature for 1.5 h, the primary antibodies against PKM1/2 (1:2000), PKM2 (1:2000), PFK (1:2000), GLO1 (1:2000), β-actin (1:2000), TPI (1:2000), LDHA (1:2000), GAPDH (1:2000), RAGE (1:1000), NF-κB P65 (1: 500), and IL-1β (1:1000) were used. β-actin was used as the loading control for general protein contents. The source-matched secondary antibodies were used, and the membranes were scanned by Odyssey 9120 (LI-COR, Inc.). Then, bands were analyzed by the Odyssey software (LI-COR, Inc.).

H9c2 cells were lysed for 20 min on ice with RIPA lysis buffer (Solarbio, Beijing, China) containing protease inhibitors and protein phosphatase inhibitor (Both purchased from Roche, Switzerland). BCA protein assay kit (Thermo, Rockford, USA) was used to detect protein concentration. Protein samples were segregated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (EMD,Millipore, USA). After blocking with 5%BSA, the blots were probed with various primary antibodies including TGF-β1, T-Smad2, T-Smad3, p-Smad2, p-Smad3, and GAPDH (1:1000 dilution, All purchased from Cell Signaling Technology, Beverly, USA) at 4°C overnight and then incubated with anti-rabbit IgG H&L (HRP) secondary antibody (1:5000, CST, Beverly, USA) for 1h at room temperature. The bands on PVDF were visualized by use of a Super Western Sensitivity Chemiluminescence Detection System (Thermo, USA). Image-J was used to analyze the band intensities.

Western blotting was performed as previously described (23 (link), 24 ). Briefly, proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing phenylmethanesulfonyl fluoride (PMSF) (Roche, Basel, Switzerland), and a protein phosphatase inhibitor (Roche, Basel, Switzerland). Thirty micrograms of proteins were subjected to 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Roche, Rotkreuz, Switzerland). The membrane was blocked with 5% (w/v) skim milk in TBST (20 mM Tris–HCl [pH 8.0], 150 mM NaCl, and 0.1% [v/v] Tween-20) for 2 h and incubated with anti-heparanase (polyclonal antibody #733), anti-iNOS (Santa Cruz Biotechnology, TX, USA), anti-p/NF-κB (Santa Cruz Biotechnology, TX, USA), anti-p/ERK1/2 (Santa Cruz Biotechnology, TX, USA), anti-phospho-p38 (Santa Cruz Biotechnology, TX, USA), anti-GAPHD (Zhongshan Golden Bridge Biotech, Beijing, China) or anti-β-actin (Zhongshan Golden Bridge Biotech, Beijing, China) as primary antibodies. The membrane was washed with TBST, incubated with horseradish peroxidase-conjugated antibody against mouse IgG (1 h at room temperature), and rinsed with TBST. Proteins were visualized with ECL Western Blotting Detection Reagents (GE Healthcare, NJ, USA) and images were analyzed with Quantity One 4.1 software (Bio-Rad). The experiments were repeated at least three times.