*2.4. Total Protein Extract and Western Blot*

LLC-PK1 cells were grown on 60 mm plates, after treatment were washed twice with cold PBS and lysed by addition of 400 μL of cold radioimmunoprecipitation assay buffer (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) with protease and phosphatase inhibitors (25 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 mM Na4P2O7, 0.5 mM glycerophosphate and 1X protease inhibitors cocktail) for 30 min at 4 ◦C with stirring. Lysates were centrifuged 15,000 × *g* during 10 min at 4 ◦C and the supernatant was collected and stored at −70 ◦C until the experiment was carried out. The protein concentration was quantified by the Lowry assay [28]. Equal amounts of protein (15 μg) were separated on 10% to 15% in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) as appropriate. Proteins were transferred onto Immobilion PVDF membranes for fluorescent application on wet transfer and blocked with 5% non-fat dry milk for 1 h. Then incubated overnight at 4 ◦C with the appropriate primary antibody: MDA (1:2000), 4HNE (1:3000), PGC-1α (1:100), NRF1 (1:200), TFAM (1:500), VDAC (1:500) OXPHOS cocktail (1:1500), MNF2 (1:1000), PINK1 (1:1000), Parkin (1:1000), LC3 (1:1500), p62 (1:1000) and α-Tub (1:8000). Then the membranes were incubated with the corresponding fluorescent secondary antibodies (1:10000) for 1 h at room temperature in darkness. Protein bands were detected in an Odyssey scanner (Li-COR Biosciences, Lincoln, NE, USA) and protein band intensity was analyzed by Image StudioTM Life Software Li-COR Odyssey, which led us to normalize to correct for loading variations between bands and give us signal counts measured in a single pixel per unit time through Z-Factor Calculation. In all cases, except OXPHOS subunits, α-TUB served as loading control. Thus, once the protein target was detected, we proceeded to detect its loading control. For that, the membranes were rinsed only with Tris-buffered saline, 0.1% Tween 20 (TBS-T 1x) 3 times for 5 min and then the membranes were incubated with primary α-TUB antibody, following the usual steps as for protein target. The OXPHOS subunits were referred to reversible Ponceau staining as loading control as described by Romero-Calvo et al. [29]. Briefly, after transfer, the membranes were incubated in 1% Ponceau S solution for 2 min. Immediately after, membranes were rinsed with PBS to remove staining saturation, clearly visible bands. Membranes were placed between transparent sheets and scanned in conventional equipment at 300 dpi as TIFF document (HP Scanjet G4050). Again, membranes were rinsed until the staining completely disappeared. Finally, membranes were blocked with 5% non-fatty milk in TBS buffer, and the usual steps were continued.
