Hpa001240

Subsequently, membranes were blocked using Li-COR Blocking Buffer (Li-COR, Lincoln, NE) for 1 hour at room temp. Mitochondrial proteins were probed for with the following primary antibodies: 3-mercaptopyruvate sulfurtransferase (3MPST, HPA001240, Sigma), mitochondrial heat shock protein 70 (mtHSP70, ab6535, Abcam), GrpE protein homolog 1 (Grpel1, sc-242966, Santa Cruz), Sirtuin 3 (SIRT3, ab86671, Cell Signaling), superoxide dismutase 2 (SOD2, sc-30080, Santa Cruz), and glutathione reductase (GR, ab16801, Abcam).Soluble proteins were probed for with the following primary antibodies: heat shock protein 70 (HSP70, ab6535, Abcam) and superoxide dismutase 1 (SOD1) (sc-11407, Santa Cruz). All primary antibodies were diluted in Li-COR Blocking Buffer at a 1:1000 ratio, with the exception of Grpel1 (1:200 ratio). Incubations were performed overnight at 4°C. Then membranes were washed in TBS-T (3 ˣ 5 min), and incubated in secondary antibody (IR Dye, 1:20,000, LI-COR) for 30 min at room temp, washed again (TBS-T, 3 ˣ 5 min), and rinsed in 1 ˣ TBS. Finally, membranes were scanned using an Odyssey Infrared Imaging System (LI-COR). For mitochondrial proteins, the immunoblot signal of each target protein was normalized to cytochrome c signals (Cyt c, sc-13156, Santa Cruz) and soluble proteins were normalized to ɑ-tubulin signals (12G10, DSHB).