Nir fluorescent secondary antibodies

The cells were lysed in the Pierce RIPA buffer (Thermo Scientific; #89900) supplemented with a protease inhibitor cocktail (Roche; #11836170001), and the protein contents of the lysates cleared by centrifugation at 12,000× g for 5 min were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific; #23225). For immunoblotting, equal amounts of protein were prepared with a Laemmli buffer (Bio-Rad; #1610747) containing 2.5% β-mercaptoethanol and separated by 4–20% polyacrylamide gel electrophoresis (PAGE) in SDS containing a Tris−glycine running buffer. The separated protein was transferred to the nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Immunoblotting was conducted using the following antibodies: mouse anti-metallothionein 1 (MT1; 1:1000; Invitrogen, Waltham, MA, USA; MA 1-25479), rabbit anti-hemoglobin-alpha (HBA; 1:1000; Proteintech, Rosemont, IL, USA; 14537-1-AP), rabbit anti-iron regulatory protein 2 (IRP2; 1:1000; Dr. Betty Leibold, University of Utah), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; Bio-Rad; 12004167) primary antibodies. The subsequent probing was processed with NIR fluorescent secondary antibodies (1:10,000; Li-Cor, Lincoln, NE, USA) and visualized using a Li-Cor Odyssey Fc detection system.

Powdered tissue samples were homogenized in a RIPA lysis buffer (Cell Signaling, Danvers, MA,) with a protease inhibitor cocktail (Roche, Basel, Switzerland). Samples were centrifuged at 16,000 g for 20 minutes and supernatant was collected. Protein concentration was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) and 30 μg of protein was loaded onto a 4-20% SDS-PAGE gel (Bio-Rad) and run at 200 V for 45 minutes. The protein was transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) at 100 V for 30 minutes using the plate electrode. Total protein normalization was assayed using a Revert 700 Total Protein Stain Kit (LI-COR, Lincoln, NE, USA) Supplementary Figures 1–4. The membrane was blocked using a blocking buffer (LI-COR) then probed with primary antibody overnight at 4° C. Primary antibodies against MTCO1 (Complex IV) (ab14705,1:2000; ab203912, 1:1000), NDUFB-8 (Complex I) (ab110242, 1:2000), and Citrate Synthase (ab129095, 1:1000) were obtained from Abcam (Cambridge, United Kingdom). NIR fluorescent secondary antibodies (LI-COR) were applied and membranes were imaged using a LI-COR Odyssey imager.