Ecl plus

Following treatment, cancer cells were lysed with RIPA buffer (0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail (HoffmannLa Roche, Basilea, Svizzera) and isolated by centrifugation at 14,000 rpm for 10 min at 4 °C. After Bradford assay quantification (Bio-Rad Hercules, CA, USA), protein lysates were subjected to SDS-PAGE and Western blot analysis.
Primary antibodies for western blot analysis against p-EGFR (Tyr1068), EGFR, p-MAPK44/42 (Thr202/Tyr204), MAPK44/42, p-AKT (Ser473), AKT, pHER2 (Tyr1221/1222), HER2, pHER3 (Tyr1289) and HER3 were obtained from Cell Signaling Technology; monoclonal anti-α-tubulin antibody (T8203) from Sigma Chemical Co. St. Louis, MO, USA. The following secondary antibodies from Bio-Rad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus (Thermo Fisher Scientific, Rockford, IL, USA) using the ChemiDoc (Bio-Rad). Each experiment was done in triplicate.

Proteins were collected and their concentrations were determined with a BCA protein detection kit (Beyotime Institute of Biotechnology, Shanghai, China). The samples (20 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were incubated in blocking solution containing 5% nonfat milk in TBST at room temperature for 1.5 h and then incubated with the primary antibodies diluted in the blocking solution individually overnight. Then, the membranes were incubated with HRP conjugated secondary antibodies (Boster Bio-engineering, Wuhan, China) for 1 h at room temperature. The bound antibodies were detected using a chemiluminescence system (ECL Plus, Thermo Scientific, and Rockford, IL).

Approximately 30 mg heart tissues collected from LV were homogenized in lysis buffer (Beyotime, Nantong, China) supplemented with a cocktail of protease and phosphatase inhibitors (Sigma Chemical Co., St Louis, MO) and centrifuged. Protein lysates from each sample were then loaded onto 12% SDS-polyacrylamide gels (40 μg/well). Following electrophoresis, separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Thermo Scientific, USA). The blotted membrane was then incubated with primary antibodies specific for AMPKα and phospho-AMPKα^Thr172 (Cell Signaling Technology, Inc., USA), glucose transporter-1 (GLUT-1) (Cell Signaling Technology, Inc., USA), glucose transporter-4 (GLUT-4) (Cell Signaling Technology, Inc., USA), PGC-1α (Abcam, UK), and β-actin (Kangcheng Biological Co., China) overnight at 4°C. After washes, blots were then incubated with horseradish peroxidase-conjugated secondary antibody (Abbkine, Inc., USA) for 2 h. Finally, immunocomplexes were visualized by ECL Plus (Thermo Scientific, USA) and analyzed with Image Lab Software (Bio-Rad Laboratories, Inc., USA). Quantification of relative protein expression was normalized to β-actin and expressed as fold change.

For frozen tissue, lysates were prepared using a RIPA lysis buffer (50mM Tris (pH7.4), 150mM NaCl, 2mM EDTA, 1% Nonidet P-40, 50mM NaF, 0.1% SDS and 0.5% sodium deoxycholate with PhosStop Phosphatase-Inhibitor Cocktail tablet and protease-inhibitor cocktail tablet (Roche). The homogenate was centrifuged at 4°C for 10 min at 14,000g and the supernatant was used for western blot. The protein content of the supernatant was quantified using bicinchoninic acid reagents and BSA standards. Equal amounts of protein were separated using a polyacrylamide SDS-PAGE gel. After SDS-PAGE, proteins were transferred to PVDF membrane. The membrane was blocked for 1 h at room temperature followed by incubation overnight at 4°C with viperin antibody (Abcam), phospho-PKCμ, phospho-ACC, phospho-Akt, or GAPDH antibodies (Cell Signaling). After overnight incubation, the blots were incubated with HRP- conjugated secondary antibodies at a dilution of 1:5,000 for 1 h at room temperature. Bands were visualized by ECL plus (Thermo Scientific) according to the manufacturer's instructions and quantified using Image lab software.

The western blot technique was performed in cerebral cortex tissue lysates, as previously described [28 (link)]. The primary antibodies used were PLP (proteolipid protein), MBP (myelin basic protein), major histocompatibility complex class II (MHC-II), CD11b (Abcam, Cambridge, UK), synaptotagmin, synapsin IIa (BD Bioscience, Madrid, Spain), Tuj-1 (Neuromics, Minneapolis, USA), and caspase 3 (active fragment of 17 kDa) (Cell Signaling, Massachusetts, USA). Membranes were washed, incubated with the corresponding HRP-conjugated secondary antibodies, and developed with the ECL system (ECL Plus; Thermo Fisher Scientific, Illinois, USA). All the membranes were stripped and incubated with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as a loading control (Chemicon, California, USA). Band intensity was quantified with the ImageJ 1.44p analysis software (National Institutes of Health, USA). The densitometry analysis is shown in arbitrary units normalized to the loading control. Additional file 1: Table S1 shows that the basal values of the various proteins evaluated by western blotting in the pup’s cortices display no significant differences when comparing both the WT and TLR4-KO groups.

Cells were treated with phloretin (50 μM) and LPS (100 ng/ml) for 1 h or 24 h to determine AMPK activation or p62, LC3 and Keap1 protein levels respectively. Cells were lysed using RIPA-buffer (150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate, 1 % SDS, 50 mM Tris), and separated via sodium dodecyl sulphate polyacrylamide gel electrophoresis. Gels were transferred to a PVDF-membrane (VWR) and blots were blocked for 1 h in 5 % bovine serum albumin in Tris-buffered saline containing 0.1 % Tween-20 (TBS-T). Membranes were probed with primary antibodies overnight at 4 °C, washed with TBS-T and incubated with the corresponding secondary horseradish peroxidase-labeled antibody for 1 h at room temperature (RT). Immunoreactive signals were detected with enhanced chemiluminescence (ECL Plus, Thermo Fisher) using the Amersham Imager 680 (GE Healthcare Life Sciences). The densities of the bands were determined using ImageJ.