Anti akt

Sample protein concentrations of cells or tissues were determined by BCA Protein Assay kit. Typically, total protein (40 μg) was electrophoresed by 8% SDS-PAGE and then transferred onto PVDF membranes. Primary antibodies used in this study were anti-NLRP3, anti-IRS (for phosphor-Ser³⁰⁷IRS), anti-IRS, anti-PI3K, anti-AKT (for phosphor-Thr³⁰⁸AKT), anti-AKT, and anti-Glut4 (all from Cell Signaling Technology, Danvers, MA, USA). After blocking, membranes were immunoblotted with primary and secondary antibodies, followed by detection with an ECL system.

HMH (Figure 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in 100% dimethyl sulfoxide (DMSO). A 50 mM stock solution of HMH was prepared and stored as small aliquots at −20 °C until needed. DMSO, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse antibodies were purchased from Sigma-Aldrich. Phospho-specific anti-p38, anti-ERK, anti-FoxO3a, anti-cdc25, anti-PI3K, anti-AKT, and anti-mTOR, and specific antibodies anti-p38, anti-ERK, anti-FoxO3a, anti-cdc25, anti-PI3K, anti-AKT, anti-mTOR, and AKT inhibitor LY294002 were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated β-actin, p53, p21, p-cdc2, cdc2, and cyclin B1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells were harvested and lysed in RIPA buffer containing a protease inhibitor cocktail (P3100; GenDEPOT, Katy, TX, USA). Blots were probed using anti-c-Raf (1:1000, Cell Signaling Technologies, 9422S), anti-phosphoAkt (Ser473) (1:1000, Cell signaling, 9271S), anti-Akt(1:1000, Cell signaling, 9272S), anti-phospho-p44/42 MAPK (Thr202/Tyr204) (1:1000, Cell signaling, 9101S), anti-p44/42 MAPK (1:1000, Cell signaling, 9102S), anti–phosphoMEK 1/2 (1:1000, Cell Signaling Technologies, 2338S), and anti-β-actin (1:3000, sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Western blot was performed as previously described (Lam et al., 2020 (link)). Specific primary antibodies [mouse monoclonal anti-human β-actin (Sigma-Aldrich); anti-PCNA, anti-PARP (Santa Cruz Biotechnology, Inc., Santa Cruz, California, United States of America); anti-PFKP, anti-LDHB, anti-Bcl-2, anti-survivin, anti-XIAP, anti-AKT, anti-CDK2, anti-CDK4, anti-CDK7, anti-Cyclin D2, H, anti-CDK4, anti-N-cadherin, anti-β-catenin (Cell Signaling Technology, Danvers, Massachusetts, United States of America); and corresponding horseradish peroxidase (HRP)-conjugated secondary (Cell Signaling Technology)] were purchased. An enhanced chemiluminescence (ECL) kit (GE Healthcare) was used to detect protein expression. Beta-actin was selected as a reference protein (Lam et al., 2020 (link)).

After 16 weeks of HFD, Mice were fasted for 6 h and then intraperitoneally administered with PBS or insulin (4 U/Kg). Mice subsequently were euthanized 15 min later, and eWAT, liver, skeletal muscle were collected, frozen in liquid nitrogen immediately, and stored at −80°C. Total protein was isolated by RIPA buffer (Beyotime, Shanghai, China) containing protease (Roche, Basel, Switzerland) and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentration was measured with a BCA kit (Dingguochangsheng, Beijing, China) and the same amount of total protein was loaded onto polyacrylamide gels. Proteins were isolated and then transferred to PVDF membranes. Membranes were blocked for 1 h in 5% BSA at room temperature. The membranes were first incubated with following anti-phosphotyrosine AKT Ser473 (1:1000, Cat#4060, Cell Signaling Technology, USA) antibody at 4°C overnight. The membranes were subsequently stripped using solution containing 62.5 mM Tris-HCl, 2%SDS, 100 mM b-mercaptoethanol at 55°C for 25 min and reincubated with anti-AKT (1:1000, Cat#9272, Cell Signaling Technology, USA) antibody at 4°C overnight. Integrated density was analyzed with Image J Software.

Soluble CD58 (sCD58), AKT inhibitor (LY294002), and Akt activator (SC-79) were acquired from Med Chem Express (MCE, Shanghai, China). Anti-Oct4 (11263-1-AP), anti-Sox2 (66411-1-Ig), anti-CD24 (18330-1-AP), anti-vimentin (10366-1-AP), and anti-c-Myc (10828-1-AP) were purchased from Proteintech (Wu Han, China). Anti-EPCAM (#2626S), anti-E-cadherin (#3195S), anti-AKT (#9272S), anti-GSK-3β (#5676S), anti-Phospho-GSK-3β (Ser9) (5558S), anti-β-Catenin (#8480S), anti-non-phosphorylated (active) β-catenin (S33/37/T41) (#8814S), anti-phospho-β-Catenin (Ser552) (#9566S), and anti-cyclin D1 (#55506S) were acquired from Cell Signaling Technology. Anti-CD58 (A0806) and anti-phospho-AKT (Ser473) (AP0140) were purchases from ABclonal (Wu Han, China).

Protein lysates were denatured and separated on 10% polyacrylamide gels. After transfer to polyvinylidene fluoride (PVDF) membranes (Roche Applied Science), membranes were blocked in 5% nonfat milk powder for one hour at room temperature and incubated with primary antibodies overnight at 4 °C. After several times of wash with TBST, membranes were incubated with secondary antibody for one hour at room temperature. Finally, the membranes were visualized using the Tanon 5200 Western blotting Detection System (Tanon, Shanghai, China). Primary antibodies used were anti-ETV6 (ThermoFisher Scientific, Waltham, MA, USA; PA5-35371, 1:1000), anti-AKT (Cell Signaling Technology, Danvers, MA, USA; 4691T, 1:1000), anti-p-AKT (Cell Signaling Technology, Danvers, MA, USA; 4060T, 1:1000), anti-p-MEK1/2 (Cell Signaling Technology, Danvers, MA, USA; 9154, 1:1000), and anti-GAPDH (Weiao, Shanghai, China; WB0197, 1:1000). For secondary antibodies, anti-rabbit IgG (Abcam, Cambridge, UK; 7074, 1:10000), anti-mouse IgG (Abcam, Cambridge, UK; 7076, 1:10000), and HRP anti-rabbit (Santa Cruz Biotech., Santa Cruz, CA, USA; 1:10000) were used. Signal was enhanced using an enhanced chemiluminescence technique (Thermo Scientific, Waltham, MA, USA; 34075).