Total oxphos cocktail

Liver samples were homogenized in RIPA buffer (Beier et ai, 2006 (link)) containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Samples were loaded onto Bolt™ 4-12% Bis-Tris gels (Invitrogen, Carlsbad, CA), followed by electrophoresis and Western blotting onto PVDF membranes (Immobilon-P Millipore, Billerica, MA). Primary antibodies against phospho(Ser2448)-mTOR (#2971, 1:1,000, Cell Signaling, Danvers, MA)phospho(Thr172)- and total AMPK (#2531, #2532, 1:1,000, Cell Signaling), and phospho(Ser473)- and total AKT (#9271, #9272, 1:1,000, Cell Signaling), and phospho(Tyr416)- and total- Src (#6943, #2123, 1:1,000, Cell Signaling), GAPDH (#sc47724, 1:2,000, Santa Cruz Biotechnologies, Dallas, TX), and total OXPHOS cocktail (#110413, 1:500, Abcam, Cambridge, MA) were used. Densitometric analysis was performed using UN-SCAN-IT gel software (Silk Scientific Inc., Orem, UT).

Western blot analysis was performed as previously described (26 (link)). Briefly, isolated mitochondrial fraction homogenates were fractionated on SDS-polyacrylamide gels and transferred to PVDF membrane. After the membranes were blocked in 5% non-fat milk, antibodies Total OXPHOS Cocktail (Cat #: ab110413; Abcam, Cambridge, United Kingdom), cytochrome c (Cat #:11940S; Cell Signaling Technology), and VDAC (Cat #:4661S; Cell Signaling Technology) were incubated at dilutions of 1:5000 overnight at 4°C in 1% TBST milk. Anti-rabbit IgG-conjugated secondary antibodies (Cell Signaling Technology) were incubated with the membranes at 1:5000 dilutions for 1 h in 1% TBST milk. Enhanced chemiluminescence developed by autoradiography was used to visualize the antibody–antigen interactions. Blots were analyzed by measuring the integrated optical density (IOD) of each band with ImageJ software (NIH, Bethesda, MD, USA).

Protein was extracted from kidney organoids cultured in EGM or REGM media for 4 days using RIPA buffer (Thermofisher) supplemented with complete protease inhibitor cocktail (Thermofisher), and centrifuged at 13,000 g for 15 mins at 4°C. The supernatant was collected, and protein concentration was measured using a bicinchoninic acid (BCA) protein quantification kit (Thermo Scientific). For western blot analyses, 25 ug of protein were separated in 10% sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and blotted onto nitrocellulose membranes. Membranes were blocked at room temperature for 1 hour with TBS 1X- 5% BSA. Membranes were then incubated in primary antibody (dilution 1:1000) Total OXPHOS cocktail (Abcam) overnight at 4°C. The membranes were then washed with PBST (PBS1X + 0.05% Tween20; Merck) for 5 minutes three times and incubated with anti-mouse secondary antibody (dilution 1:10,000) (IRDye®680RD Goat anti-Mouse; LI-COR). After washing with PBST for 5 minutes twice and with PBS for 5 minutes once, membrane-bound antibodies were detected by fluorescence with the Odyssey ® Fc Imaging System. Alpha-tubulin (1:5000; Sigma) was used as a loading control for normalization and quantification. Images were analyzed with Image Studio Lite Version 5.2 software.

Protein lysates were prepared from frozen powdered liver using T-PER buffer (ThermoFisher #78510) containing protease inhibitor cocktail and phosphatase inhibitors from Sigma-Aldrich (St. Louis, MO). Western blot analyses were performed using standard PAGE-SDS as previously described (25 (link)). Primary antibodies were as follows: ACC (#3676), p-ACC (#11818), AMPKα (#5831), p-AMPKα (#2535), AMPKβ½ (#4150), p-AMPKβ1 (#4181), and Pex5 (#83020) from Cell Signaling; Catalase (ab16731), Total OXPHOS cocktail (ab110413), Pex19 (ab137072), and PMP70 (ab3421) from Abcam; GLUT2 (07–1402) and Pex14 (ABC-142) from EMD Millipore, and PGC-1α (sc-517380) from Santa Cruz Biotechnology. HRP-linked secondary antibodies (anti-mouse IgG Cat# NXA931 and anti-rabbit IgG Cat# NA934V) were purchased from GE Healthcare (Piscataway, NJ) and proteins were detected using ECL chemistry. Reversible protein stain-MemCode (MemC) (Thermo-Fisher #24580) was used to confirm equal transfer of proteins and quantitation of these bands served as a loading control. A ChemiDoc imaging system (Bio-Rad) was used to image bands and Image Lab software (Bio-Rad) was used to quantitate band intensity. Data are expressed as fold change compared to the Con-Sed group after normalizing to MemC.