Hexokinase II

Western blots were performed as previously described on freshly isolated hearts (i.e. separate from the perfused hearts) [11 (link),18 (link)]. Briefly, fifty micrograms of total protein extract from mouse heart tissue was separated electrophoretically and transferred to PVDF membrane. The membranes were probed with the following antibodies: pyruvate kinase M2 isoform (PKM2, #4053), lactate dehydrogenase A (LDHA, #2012), acetyl-CoA carboxylase (ACC, #3662), phospho-acetyl-CoA carboxylase, (p-ACC, Ser 79, #3661), and pyruvate dehydrogenase (PDH, #2784) were purchased from Cell Signaling Technology, Inc. (Danvers, MA); hexokinase II (HXKII, sc-6521), glutamine-fructose-6-phosphate transaminase 2 (GFAT2, sc-134710), O-GlcNAcase (OGA, sc-135093) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α, sc-13067) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); O-GlcNAc transferase (OGT, O6264) from Sigma-Aldrich Corp. (St. Louis, MO); phospho- pyruvate dehydrogenase (p-PDH, Ser293, #ABS204) was purchased from EMD Millipore Corporation (Billerica, MA);malonyl CoA-carboxylase (MCD, 15265-1-AP) was purchased from Proteintech (Chicago, IL); glucose transporter 4 (Glut4, GTX88031) was purchased from GeneTex (Irvine, CA). Muscle carnitine palmitoyltransferase 1 (M-CPT1) and pyruvate dehydrogenase kinase (PDK)2 and PDK4 were obtained as a personal gift from Gebre Woldegiorgis (Oregon Health Sciences University, Beaverton, OR) and Robert Harris (Indiana University School of Medicine, Indianapolis, IN). Total protein O-GlcNAc levels were determined using an antibody (RL-2) from Abcam (Cambridge, MA) on freshly isolated protein. Western blots were visualized with enhanced chemiluminescence upon exposure to Kodak BioMax Light ML-1 film. Membranes were stripped by washing for 30 min with 100 mM dithiothreitol, 2% (wt/vol) SDS, 62.5 mM Tris·HCl, pH 6.7, at 70°C, followed by three 10-min washes with TBS for additional antibody analysis. Immunoblots of proteins without a phosphorylated form were normalized to total protein staining in the molecular weight region around the band of interest by Thermo Scientific Pierce Reversible Protein Stain Kit for PVDF Membranes (Thermo Scientific, Rockford, IL), which are shown. Phosphorylated proteins were normalized to their appropriate total protein.

Measurements of non-structural carbohydrate (NSC) were performed on 1 cm² leaf discs, collected from leaf lamina with a cork borer, and immediately frozen in liquid nitrogen. Soluble carbohydrates were extracted at 80 °C for 45 min, after grinding the leaf discs in a glass-glass homogeniser, containing 1.5 mL of 80% (v/v) ethanol, 20% (v/v) 100 mM Hepes (pH 7.1), and 10 mM MgCl₂. After centrifugation at 16,000× g for 5 min, the supernatant was analysed for soluble carbohydrate content. Glucose, fructose, and sucrose were determined by an enzymatic coupled spectrophotometric assay with hexokinase (1.2 U), glucose phosphate dehydrogenase (0.3 U), phosphoglucose isomerase (0.3 U), and acid invertase (30 U) as described in Moscatello et al. [43 (link)].
Starch was quantified from the pellet remaining after extraction of soluble sugars, after three washing with 50 mM NaAcetate buffer (pH 4.5), suspended in 1 mL of the same buffer, and autoclaved at 120 °C for 45 min. The suspension was incubated at 50 °C for 1 h with amyloglucosidase (40 U) and α-amylase (4 U). The glucose produced by starch hydrolysis was determined enzymatically as described above.

Western blotting analysis was applied to examine protein levels using antibodies recognizing Rho A (Abcam, Cambridge, MA, USA), ROCK1 (Abcam), ROCK2 (Abcam), myosin phosphatase-targeting subunit 1 (MYPT1) (Cell Signaling Technology (CST), Danvers, MA, USA), phosphorylated (p)-MYPT1 (CST), glucose transporter type 1, erythrocyte/brain (GLUT1) (Abcam), hexokinase 2 (HK2) (Abcam), pyruvate dehydrogenase kinase 1(PDK1) (Abcam), phosphofructokinase 1 (PFK1) (CST), lactate dehydrogenase A (LDHA) (Abcam), osteopontin (OPN) (Abcam), RUNX2 (Abcam), AMPK (Abcam), p-AMPK (Abcam), ubiquitination (Proteintech, Rosemont, IL, USA), Sodium Potassium ATPase (Abcam), and β-actin (Proteintech). Human aortic valve tissues and VICs were rinsed with cold 1× PBS, and then lysed on ice in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotech; 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM Ethylenediaminetetraacetic acid (EDTA), and leupeptin) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) for 15 min, followed by homogenization with 20 kHz ultrasonic lapping and centrifugation at 14,000 rpm at 4 °C in a refrigerated microcentrifuge for 15 min to extract total protein. Then, supernatants were stored and a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime Biotech) was used to detect the total protein concentration to normalize the samples. Equal amounts of samples (30–45 μg/lane) were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to polyvinylidene difluoride (PVDF) membranes using a wet-transfer system. The membranes were blocked using QuickBlock Blocking Buffer (Beyotime Biotech) at room temperature for 20 min, and then incubated with primary antibodies at 4 °C overnight. After three washes with Tris-buffered saline-Tween-20 (TBST) (Servicebio, Wuhan, China), we incubated membranes with the corresponding secondary antibodies coupled with horseradish peroxidase (HRP) for 90 min on shakers. After three washes with TBST, enhanced chemiluminescence (ECL) signals (Amersham Biosciences, Piscataway, NJ, USA) were detected using an ECL kit (Millipore, Billerica, MA, USA) with Imaging system (Thermo Fisher Scientific). Densitometric quantification was performed using Image J software.