Cytochrome c release assay kit

For proteins isolation, cells were lysed in RIPA buffer and quantified using Pierce BCA kit (Thermo-Fisher). For cytosolic/mitochondrial fractionation Cytochrome c Release Assay Kit (abcam, ab65311) was used. Proteins lysates (10–20 μg) were resolved on 5%-12% SDS–PAGE gels and transferred to PVDF membrane (Thermo-Fisher). Membranes were blocked in 5% Milk (BioRad) or 5% BSA (Sigma) in 1X TBST and incubated overnight at 4 °C in primary antibodies. Membranes were then washed with 1X TBST and incubated with secondary antibodies (Southern Biotech) for 1 h. The membranes were developed with ECL reagent (Thermo Fisher) on to X-ray films (Thermo-Fisher) using the chemiluminescence imager, AGFA CP100. Rabbit anti-HSPD1 (ab46798, 1:20,000) and rabbit anti-NLRC5 (ab117624, 1:1000) antibodies were purchased from Abcam; mouse anti-TOMM20 (H00009804-M01, 1:1000) was purchased from Abnova; anti-β-Actin (8H10D10) HRP conjugate (1:10,000) was purchased from Cell Signaling. Protein band density was quantified using ImageJ.

Cytochrome c Release Assay Kit (ab65311, Abcam) protocol was followed. Briefly, after the mitochondria isolation, cytosolic and mitochondrial fractions of protein lysates of the same samples were run on the 4–12% Bis-Tris gel; proteins were separated and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA followed by incubation overnight at 4 °C with anti-Cytochrome c mouse antibody (1 μg/mL) prepared in 5% BSA. In order to further confirm the mitochondrial isolation efficiency, membranes were incubated with COXIV Mouse Antibody (ab14744, Abcam) at 1:1,000 dilution prepared in 5% BSA overnight at 4 °C. After washing, membranes were incubated for 1 h at room temperature with appropriate secondary antibodies (HRP-conjugated; dilution 1:5,000). Prestained molecular weight markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with an enhanced chemiluminescent reagent, and the signals were monitored on Amersham imager 680 (GE Healthcare Bio-Sciences Corp.). Relative band intensity was determined by densitometry using Image-J and normalized with β-actin protein.