Anti hk2

Evaluation of protein expression was performed as previously described (11 (link), 28 (link)). Briefly, frozen muscle samples were homogenized in sucrose buffer pH 7.4 (10 mmol/L Tris–HCl, 1 mmol/L EDTA and 250 mmol/L sucrose), centrifuged at 760 g for 10 min at 4°C, and the supernatant was used as a total cellular protein fraction. Protein concentration was determined by Bradford method (Bio-Rad Laboratories, Hercules, CA, USA).
Equal amounts of protein (20–50 μg, according to the target protein) were electrophoresed, transferred to nitrocellulose membrane and immunoblotted with anti-GLUT4 (1:3500, EMD Millipore, Billerica, MA, USA, #07-1404), anti-HK2 (1:1,000, Cell Signaling Technology, Boston, MA, USA, #2867S), anti-NFKB1 (1:1000, Cell Signaling Technology, Boston, MA, USA, #12540S) or anti-RELA (1:450, Abcam, Cambridge, MA, USA, #7970). The membranes were incubated with appropriate secondary conjugated antibody, according to manufacturer's specifications, and signal was detected by enhanced chemiluminescence procedure. The optical density of the blots was quantified by densitometry (ImageScanner III, GE Healthcare, Uppsala, Sweden) and normalized by the densitometry of the respective lane measured in the Ponceau S stained membrane (29 (link)). Results were expressed as arbitrary units (AU) per μg of protein, and considering the mean of control values as 100.

Cells were collected and lysed using RIPA buffer (150 mM NaCl, 1% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris-HCl pH 8) supplemented with protease and phosphatase inhibitors (Roche, Switzerland) and total protein concentration were measured by BCA assay. Whole-cell lysates (~20–30 μg of proteins) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblotting was performed using the following primary antibodies: anti-p38 (#8690), anti-phospho p38 (T180/Y182 - #4511), anti-p53 (#2524), anti-phospho pS6 (S235/236 - #4058), anti-Akt (#9272), anti-phospho-Akt (S473, #4060), anti-caspase-3 (clone 8G10; #9665), anti-HK2 (#2867), anti-HER2 (#2165), anti-mTOR (#2983), anti-phospho-mTOR (S2448—#5536), anti-PARP (#9542) from Cell Signaling Technology (Massachusetts, USA); anti-LC3 (L7543) from Sigma-Aldrich; anti-Actin (sc-8432), anti-GAPDH (sc-166545), anti-IκB-α (sc-371), anti-Lamp2 (sc-18822) and anti-tubulin (sc-73242) from Santa Cruz Biotechnology (Texas, USA); and anti-Hsc70 (10654–1-AP) from Proteintech (Illinois, USA).

Chrysin and other chemical reagents such as Tris, NaCl, SDS and DMSO were purchased from Sigma-Aldrich (St. Louis, MO). The normal human hepatic cell LO2 and HepG2, Hep3B, Huh-7, HCC-LM3, Bel-7402 and SMMC-7721 were obtained from the Cell Bank of Chinese Academy of Sciences and cultured with Dulbecco’s Modified Eagle Medium containing 10% FBS and 1% antibiotics in a 37 °C incubator with 5% CO₂. Anti-HK2, anti-VDAC1, anti-cleaved-caspase3, anti-cleaved-PARP, anti-cytochrome C, anti-Bax, anti-Bak, anti-Bcl-2, anti-Bcl-xL, anti-rabbit IgG-HRP and anti-mouse IgG-HRP antibodies were products of Cell Signaling Technology, Inc. (Danvers, MA). Anti-α-Tubulin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (A5316) was product of Sigma (St. Louis, MO, USA). HK2 (ORF004940) construct was purchased from Applied Biological Materials (ABM) Inc. (Richmond, BC, Canada). Lipofectamin 2000 was purchased from Invitrogen (Carlsbad, CA).

Samples were lysed in 200 μL RIPA buffer (pH 7.4) containing protease inhibitors (ThermoFisher Scientific). 20-25 μg protein suspended in SDS loading buffer was run on 10% SDS polyacrylamide gels and electrotransferred to PVDF membranes. Membranes were blocked in 5% milk and incubated with 1:1,000 dilutions of primary antibodies in 5% BSA, including anti-HK1 (sc-#46695, Santa Cruz Biotechnology, Dallas, TX, USA), anti-HK2 (Catalog #2867, Cell Signaling Technology, Danvers, MA, USA), anti-integrin β4 (Cell Signaling Technology, Cat# 4707) and the loading controls anti-β-Actin (sc-#47778, Santa Cruz Biotechnology) and anti-vinculin (Catalog #V9131, Sigma). Membranes were incubated with 1: 5000 dilutions of appropriate secondary antibodies in 5% milk (ThermoFisher Scientific). Incubations were for 1 hour at room temperature and Clarity Western ECL substrate with ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA, USA) were used to detect immunoreactive bands.

The Flag-CTL and Flag-GDPD5 plasmids were transfected into SH-SY5Y cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), as well as anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).

The GFP-CTL and GFP-HMGCS2 plasmids were transfected into OSRC2 cells with Neofect™ DNA transfection reagent. After cells were transfected for 48 h, the cells were lysed by 1% SDS lysing buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail (Apexbio, Houston, TX, USA) for Western blot analysis. The protein concentration was determined by a BCA protein assay reagent kit (Thermo Scientific, Waltham, MA, USA). All blots were, respectively, incubated with primary anti-bodies anti-flag (1:1000, ABclonal, Wuhan, China), anti-p-ACC, anti-ACC, Anti-p-ACLY, anti-ACLY, anti-PFKFB3, anti-HK2 and anti-LDHA (1:1000, Cell Signaling Technology, MA, USA), anti-ALDOA, anti-ENO2, anti-HADH and anti-PPARA (1:1000, proteintech, Wuhan, China), and anti-β-Tubulin (1:5000, TRANSGEN, Beijing, China). Bands were visualized with ECL Reagents (Smart-Lifesciences, Changzhou, China).