Pierce 660 assay

The rat cell lines B35, H19-7/IGF-IR, RN33B and PC12 were obtained from the ATCC (Manassas, VA). Cells were grown in DMEM-F12 containing 10% FBS and 1% penicillin/streptomycin. RN33B and H19-7/IGF-IR cells were grown at 33° C, while B35 and PC12 cells were grown at 37° C. Cells were harvested and mitochondria were isolated by sequential differential centrifugation (Mitosciences, Eugene, OR) followed by an immunomagnetic (anti-TOM22) affinity isolation (Miltenyi Biotech, Auburn, CA) [11 ]. Mitochondria were lysed in 4% sodium dodecyl sulfate (SDS) and protein concentration was quantified using a using a Pierce 660 assay with bovine serum albumin standards (Thermo Fisher Scientific, Rockford, IL).

THP-1 macrophages were washed thrice with phosphate-buffered saline (PBS, HyClone), and collected for either protein or RNA. To obtain protein from whole cell lysates, cells were scraped in PBS and pelleted by centrifugation at 1000 x g for 5 min at 4°C and re-suspended in 300 μL per 10⁶ cells of lysis buffer. Lysis buffer contained 2% (w/v) SDS (Bio-Rad; Hercules, CA), in 0.1 M Tris-HCl (Fisher Scientific), 0.1 M DTT (Promega; Madison, WI) and 1X Protease Inhibitor Cocktail (Sigma-Aldrich). Cells were heated at 95°C for 5 min, allowed to cool and, 100 units/mL Benzonase Nuclease (Merck KGaA; Darmstadt, Germany) were added. Protein was quantified using Pierce 660 Assay as per the manufacturer's protocol using 50 mM ionic detergent compatible reagent (Thermo Scientific; Rockford, IL). Twenty micrograms of whole cell lysate from each condition were loaded onto a 4-12% bis-tris gel (Life Technologies) and separated at 100 V for 90 min and transferred to a PVDF membrane at 25 V for 90 min. Membranes were blotted with antibodies recognizing the following antigens: NF-κB/p65 [Cat# 8242] (New England Biolabs, Ipswich, MA), TLR9 [Cat# ab85860] (Abcam, Cambridge, United Kingdom), and IRAK1 [Cat# ab238] (Abcam) all at 1:1000 concentration overnight in 4°C.