Anti na k atpase

H&E and Masson staining, immunohistochemical and immunofluorescence staining were performed according to an established procedure. The following primary antibodies for immunohistochemical and immunofluorescence staining were used: anti-Acetyl-lysine (cat: 9441, CST), anti-SIRT3 (cat: 2627, CST; cat: 365175, Santa Cruz), anti-Na/K-ATPase (cat: sc-28800, Santa Cruz), anti-FN (cat: F3648, Sigma Aldrich), and anti-Collagen I (cat: 1310-01, Southern Biotech).

The cells were washed with precold PBS three times, and total and fractionated cellular proteins were isolated. The cytoplasmic and nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN Biotech). The membrane protein was extracted using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After protein content determination, extracted proteins were separated by using 10% SDS-PAGE gel and then transferred onto nitrocellulose membranes using a Bio-Rad transfer blotting system. The membranes were incubated with primary antibodies against ABCG1 (Proteintech, USA), Nox4 (Santa Cruz Biotechnology, USA), HO-1 (Santa Cruz Biotechnology, USA), Nrf2 (Cell Signaling Technology), and p47phox (Santa Cruz Biotechnology, USA) antibodies. Proteins were visualized using an enhanced chemiluminescence detection system (ECL, Cell Signaling Technology Inc.). Anti-β-actin, anti-lamin B1, anti-Na+/K+ATPase (Santa Cruz Biotechnology, USA) were used to control for equal protein loading.

Localization of ROS-GC1 WT and mutants in HEK cells by immunofluorescence microscopy was done as described before using an Olympus fluorescence microscope (Zägel et al., 2013 (link)). The following primary antibodies were used for detection: anti-ROS-GC1 (1:100; ROS-GC1 (H-225), sc50512, rabbit polyclonal IgG; Santa Cruz Biotechnology), anti-Na⁺/K⁺-ATPase (1:200; Na⁺/K⁺-ATPase, a (H-3), sc-48345, mouse monoclonal IgG_2b; Santa Cruz Biotechnology), and anti-calnexin [1:200; calnexin (E-10), sc-46669, mouse monoclonal IgG_2a; Santa Cruz Biotechnology]. Secondary antibodies were donkey anti-rabbit conjugated to Fura350 (dilution 1:200) from Invitrogen and goat anti-mouse conjugated with Dylight594 from Thermo Scientific, USA used in a dilution of 1:500. Incubation and washing buffers were exactly as described (Zägel et al., 2013 (link)).

Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitors. Homogenates were maintained in ice for 30 min and centrifuged at 15 000 × g for 10 min at 4 °C, and the supernatant was recovered. Protein concentration was determined by BCA protein assay kit (Life, New York, NY, USA). Proteins were resolved in SDS-PAGE (10% polyacrylamide), transferred to PVDF membrane, and incubated with primary antibodies. The reactions were followed by incubation with peroxidase labeled secondary antibodies (Life). Primary antibodies used were: anti-RIPK1 (1:800, Abcam), anti-RIPK3 (1:800; Abcam), anti-MLKL (1:1000, Abcam), anti-β-actin (1:5000, Abcam), anti-Na+-K+-ATPase (1:400; Santa Cruz, Santa Cruz, CA, USA), anti-CaMKIIδ (1:800, GeneTex), anti-p-CaMKII (1:800, Thermo), and anti-ox-CaMKII (1:600; Millipore, Bedford, MA, USA). CaMKII activation was assessed by measuring phosphorylation and oxidation levels.

Total protein extraction and Western blot analysis were performed, as previously discussed [67 (link), 68 (link)]. For MCT4 analysis, cell membrane proteins were isolated using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Fisher Scientific) according to the manufacturer’s protocol. Different ice-cold buffers were employed to sequentially separate cytoplasmatic, membrane, soluble nuclear, chromatin-bound nuclear, and cytoskeletal extracts. The following antibodies were used: anti-MCT4 (Santa Cruz Biotechnology and GeneTex sc-376140), anti-GLUT-1 (Santa Cruz Biotechnology sc-377228), anti-Na⁺/K⁺ ATPase (Santa Cruz Biotechnology, sc-48345), and anti-β-actin (Sigma-Aldrich- clone AC-15, ascites fluid). Secondary antibodies were goat anti-rabbit and anti-mouse IgG (ImmunoReagents, INC GtxRB-003-DHRPX, GtxMu-003-DHRPX). Protein levels were quantified by densitometric analysis via ImageJ software. Fold change was calculated over control.