Anti glut4

After immunoblotting, the intensity of the immunoblot bands was detected utilizing a ChemiDoc XRS+ (Bio-Rad) instrument. Antibodies against the following proteins were purchased from Cell Signaling Technology: anti-Bax (1:1,000), anti-Bcl-2 (1:1,000), anti-GSK3beta (1:1,000), anti-phosphorylated GSK3beta (1:1,000), anti-AKT (1:1,000), anti-p-AKT (Ser473; 1:1,000), anti-pThr172-AMPKα (1:1,000), anti-GLUT4 (1:500) and anti-GAPDH (1:10,000; loading control) antibodies. In order to measure the amount of GLUT4 in the plasma membrane, the plasma membrane protein was separated from the cell by using a compartmental protein extraction kit (Millipore, USA).

For the western blot analysis, 50 mg of LV myocardial tissue was homogenized on ice in radioimmunoprecipitation lysis buffer containing a complete protease inhibitor cocktail (Roche Diagnostics). The homogenates were centrifuged to collect the supernatants. The protein concentration was determined using a BCA protein assay kit (Thermo Scientific). LV myocardial samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA in PBS plus Tween 20 at room temperature for 2 h, and then incubated with anti-PI3K (P110α), anti-Akt (Ser172), anti-phospho-Akt (Ser172), anti-GLUT4, anti-AMPK (Thr172), anti-phospho-AMPK (Thr172), and anti-eNOS antibodies (Cell Signaling Technology, Danvers, MA) at 4 °C overnight. Membranes were subsequently washed and incubated with the relevant secondary horseradish peroxidase-conjugated antibodies (1:5000; ZSGB-BIO, Beijing, China) for 90 min. β-actin (mouse anti-β-actin; 1:500; ZSGB-BIO) was used as the loading control. Intensity was measured using Quantity One densitometric software (Bio-Rad Laboratories, Hercules, CA), as described previously [18 (link)].

The following primary antibodies were used for immunoblots: Anti-Hexokinase II (Cell Signaling Technology, 2867), Anti-GLUT1 (Cell Signaling Technology, 12939S), Anti-GLUT4 (Cell Signaling Technology, 2213S), Anti-CPT1β (Abcam, ab134988), Anti-VLCAD (Abcam, ab155138), Anti-Acadm (Santa Cruz, sc365108), Anti-PPARα (Cayman, 101710 and Novus Biologicals, N300-537), Anti-PGC-1α (Millipore, Ab3242), Anti-GAPDH (Cell Signaling Technology, 2118C), Anti-Tubulin (Cell Signaling Technology, 3873S). Secondary antibodies used were Anti-Rabbit IgG, HRP-link (Cell Signal Technology, 7074S) and Anti-Mouse IgG, HRP-link (Cell Signal Technology, 7076S). In all Western blot images shown in figures, individual lanes correspond to individual mice. Molecular weights (MW) are shown. The signal intensity of Western blots was quantified using ImageJ software and then normalized by the corresponding loading control (Tubulin or GAPDH).

Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.

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.

Crude extracts from dissected muscle or heart of experimental animals were washed twice with ice-cold PBS and lysed in RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% SDS, and 0.5% deoxycolate), supplemented with a cocktail of protease inhibitors (Sigma) and phosphatase inhibitors (Thermo Scientific). Heart, muscle lysates or serum samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore). The blots were washed with Tris-buffered saline Tween-20 (TBST), blocked with 5% milk in TBST for 1 h, and incubated with custom-made monoclonal anti-MG53 antibody (1:2000)^{18 (link),30 (link)} or commercial IRS-1 antibody (Invitrogen, Cat. No. 700662, 1:1000), anti-PPARα antibody (NovusBio, Cat. No. NB600-636, 1:1000), anti-IR-β antibody (Cell Signaling, Cat. No. 3025, 1:1000), and anti-Glut-4 (Cell Signaling, Cat. No. 2213, 1:1000) antibody. Immunoblots were visualized with an ECL plus kit (Pierce).
We routinely used multiple samples derived from the samples in a given western blot. To illustrate the spread of the band intensity with the wild type samples, we normalized the band intensity to one wild type sample (which is often the one with middle intensity), and plotted the relative intensity of other samples (including wild type and tPA-MG53 muscles) in the scatter plot.