Myecl imager system

In total, 15 μg and 10 μg of proteins of hippocampus and BAT homogenates, respectively, were loaded and separated by SDS-PAGE, as previously described [17 (link)]. The list of antibodies used in this study is available in Additional File 3. Homogenates were all run on the same gel for each experiment. Membranes were imaged using the myECL imager system (Thermo Fisher Scientific). Quantifications were performed using the ImageLab software (Millipore), and the results were expressed as relative optical densities (OD). For the analysis of the protein tau, bands from all isoforms detected around 60 kDa were selected and quantified together.

Western blotting of PBMC whole cell lysates was performed according to standard protocols using the XCell Sure Lock™ Mini-Cell Electrophoresis System. Equalization of protein concentrations of the whole-cell lysates was performed using the BCA Protein Assay Kit according to the manufacturer´s instructions (both from Thermo Scientific, Rockford, USA).
HRP-conjugated rabbit monoclonal anti-GAPDH was used as a loading control (conc. 1:10.000, 4 °C overnight incubation, Thermo Scientific, Rockford, USA). The primary antibodies used were rabbit monoclonal anti HO-1, mouse monoclonal anti-NQO1 and rabbit monoclonal anti-SOD2 (Cell signaling technologies, Danvers, USA; each diluted 1:1.000). Visualization of immunoreactivity was carried out by HRP-conjugated secondary antibodies (each diluted 1:10.000, DAKO A/S, Glostrup, Denmark), Super-Signal West Pico chemoluminescence (Pierce, Rockford, USA) and subsequent digital image acquisition using the myECL™ Imager system (Thermo Scientific, Westham, USA). We performed densitometry using ImageJ software, v. 1.51 (NIH, Bethesda,USA) and calculated greyscale value ratios with DMSO control after normalizing protein intensity ratios with GAPDH from the identical experiment as a reference.

The XCell Sure Lock™ Mini-Cell Electrophoresis System served as a platform for western blot experiments according to standard protocols. For equalization of protein concentrations, we used the BCA Protein Assay Kit (both from Thermo Scientific, Westham, United States). Amounts of HO-1 were detected using primary rabbit monoclonal anti-HO-1 (dilution 1:1,000, Cell Signaling, Danvers, United States) and secondary HRP-conjugated polyclonal goat anti-rabbit (dilution 1:10,000, Thermo Scientific, Westham, United States). For digital image acquisition we used the myECL™ Imager system (Thermo Scientific, Westham, United States). For calculation of HO-1/GAPDH-ratios greyscale intensity values were determined by ImageJ software, v. 1.51 (NIH, Bethesda, United States).

An aliquot of cell extract (10 µg) was added to each well, and subjected to SDS-PAGE under reducing conditions. The separated proteins were then transferred to polyvinylidene fluoride membranes for 30 min at 15 V. After blocking in TBS-Tween-20 (0.1%) buffer with 5% skim milk for 2 h at room temperature, the membranes were incubated at 4 °C overnight with an anti-lumican antibody (1:1000; cat. no. MAB2846; R&D Systems, Inc., Minneapolis, MN, USA). Next, the membranes were washed and incubated at room temperature for 1 h with HRP-conjugated anti-mouse IgG antibody (cat. no. A102PU; American Qualex, San Clemente, CA, USA). The blots were washed and visualized using SuperSignal West Dura Extended Duration substrate (Thermo Fisher Scientific, Inc., Rockford, IL, USA), with band detection using the myECL Imager system (version 2.0; Thermo Fisher Scientific). Finally, the same membranes were re-probed using an anti-β-actin antibody (cat. no. sc-47778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), which served as the protein loading control. All Western blots were performed in three independent experiments.

Lysis, immunoprecipitation and immunoblotting were performed as previously described [38 (link)]. For subcellular fractionations, cytoplasmic and nuclear extracts were obtained using the Subcellular Protein Fractionation Kit, according to the manufacturer’s instructions (Thermo Scientific). Cell lysis and purification of Ubiquitin conjugates was performed as described in Moretti et al. [70 (link)], at room temperature in denaturing conditions (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-Hcl pH8, 1% Triton X-100 and 20 mM Imidazole). The chemiluminescence reaction was visualized and processed using myECL Imager system (Thermo Scientific). For relative quantification, densitometry analyses were performed using myImageAnalysis Software (Thermo Scientific). Band intensities in the linear range of the signal were measured using underexposed membranes. Two different concentrations of each sample were tested and samples with lower concentrations were used for analyses.

The cell extract, culture medium, and commercial human normal serum (ImmunoBioScience Corp., Mukilteo, WA) were subjected to SDS-PAGE under reducing conditions. The separated proteins were transferred to polyvinylidene fluoride transfer membranes. Membranes were incubated with an anti-annexin A2 rabbit monoclonal antibody, anti-aldolase A rabbit monoclonal antibody, or anti-cyclophilin A antibody (Cell Signaling Technology Inc., Beverly, MA, USA) at 4 °C overnight. Membranes were then washed and incubated with HRP-conjugated anti-rabbit IgG antibody (American Qualex, San Clemente, CA). After washing, blots were visualized by enhanced chemiluminescence and detected using a myECL Imager system (ThermoFisher Scientific). The same membranes were reprobed with anti-β-actin antibody (Sigma) to confirm equal loading of the proteins. All Western blot analyses were performed three times.