Image studio lite ver 5

Tissues and cells were lysed in 50 mM Tris-base-HCl, 150 mM NaCl and 1% NP40 (I8896-50ML, Sigma Aldrich, Overijse, Belgium) buffer. Protease and phosphatase (11836153001 and 04906837001, Sigma Aldrich, Overijse, Belgium) inhibitors were added just before lysis. Samples were maintained on ice during the procedure. Cell debris were pelleted by centrifugation (14,000× g, 10 min, at 4 °C). Proteins were quantified using a Bradford assay (23200, Thermo Fisher Scientific, Merelbeke, Belgium). Samples containing 30 to 50 µg of total proteins were electrophoresed on 7.5% to 12.5% SDS polyacrylamide gels. PVDF membranes were blocked with a solution of 5% low-fat milk diluted in Tris-buffered saline (TBS)/0.1% Tween-20. Membranes were incubated overnight with primary antibodies, at 4 °C. The used antibodies are listed in Table 1. The next day, membranes were washed with TBS/0.1% Tween-20 and incubated with the secondary antibodies for 1 h, at RT. After incubation, membranes were washed again and revealed using chemiluminescence (kits 34577 and 34094, Thermo Fisher Scientific, Merelbeke, Belgium). Pictures were taken with a Fusion Solo S machine (Vilber, Collégien, France). Densitometric analysis was performed using the Image Studio Lite Ver 5.2 software (LI-COR Biosciences, Lincoln, NE, USA).

Western blot analyses of whole-cell protein lysates (RIPA lysis buffer supplemented with protease and phosphatase inhibitors, Thermo Fisher Scientific) were performed as described^{12 (link)}. Protein concentrations were assayed using a BCA protein assay Kit (Pierce). Equal amounts of protein were separated by SDS-PAGE using 4–20% gradient Tris-glycine gels (Bio-Rad) and transferred to nitrocellulose membranes (Bio-Rad). After incubation with primary and secondary antibodies, the proteins were detected using Western Bright Quantum chemiluminescence detection system (Advansta). Scanning of gels was performed using Epson Perfection 4490 Photo Scanner, and quantification using Fiji ImageJ. In some cases, Odyssey imaging (Li-Cor Biosciences) was used and the relative band densities quantified with Image Studio Lite Ver 5.2 software (Li-Cor Biosciences).

Cells were washed with phosphate-buffered saline (PBS) and protein was harvested with lysing buffer (20 mM Tris-HCl [pH 7.8], 150 mM NaCl, 1 mM sodium orthovanadate, 1% Igepal, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin), as described (Crupi et al., 2020 (link); Hyndman et al., 2017 (link)). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes, as previously described (Crupi et al., 2020 (link); Hyndman et al., 2017 (link)). Blots were probed with indicated primary (1:1000–1:5000 dilution) and secondary IRDye antibodies (1:20,000) and visualized using the Odyssey DLx Imaging System (LI-COR Biosciences). Immunoblot band intensity quantification was performed using ImageStudio Lite Ver 5.2 software (LI-COR Biosciences).

Kidney and liver lysates were prepared from frozen tissues, homogenized, centrifuged, proteins quantitated, and samples electrophoresed and transferred to nitrocellulose membranes, as described previously (Pastor-Soler et al., 2022 (link)). The membrane was first stained with Revert 700 total protein stain solution (LI-COR) and then washed prior to imaging at 700 nm for total protein quantitation per lane, as per the manufacturer’s recommendations. After destaining, the membrane was then blocked and probed with primary and secondary antibodies of interest. Quantification of immunoblot images was performed by densitometry and analyzed using Image Studio Lite Ver 5.2 software (LI-COR, United States). Please refer to Table 1 for information on antibodies and blotting conditions.