Carbonyl cyanide m chlorophenyl hydrazone cccp c2759

For mitochondrial oxidant measurement and mitochondrial membrane potential (ΔΨm), Flp‐In™ T‐REx™ 293T Flag and Flag‐NEK5^WT cells were plated in 12‐well plates 72 h before for protein expression induced by tetracycline. On the day of the experiment, probes were added to complete culture medium. MitoSOX Red (Thermo Scientific, # M36008) was added at 5 µm for 30 min and was simultaneously incubated with 0, 0.5, 1, or 2 µm of antimycin A. For ΔΨm detection, tetramethylrhodamine, ethyl ester, and perchlorate (TMRE) (Thermo Fisher Scientific, T669) were utilized at a final concentration of 200 nm, with carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; C2759; Sigma‐Aldrich) at a final concentration of 10 μm used as positive control for 30 min. After the incubation, cells were washed once with PBS, trypsinized, and resuspended in 500 μL of PBS containing 2% FBS. The analysis was performed immediately in a BD FACS Verse. Median fluorescence intensity (MFI) from nonstained samples was used to normalize each cell, and normalized MFI from control, Flp‐In™ T‐REx™ 293T Flag cells, was set as 100%. The graph represents the relative fluorescence of the NEK5^WTcells to control.

Cortisol, corticosterone, BSA, DAPI, α-tubulin antibody (T6074), melatonin (M5250), PD98059 (027K2176), 5-azacytidine (5-aza, A2385) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP, C2759) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Lamin A/C (sc-376248), β-actin (sc-47778), MT2 (sc-28453), dynein (sc-13524), NUP62 (sc-48373), importin β (sc-137016), BNIP3 (sc-56167), c-MYC (sc-40), IgG mouse (sc-2025), p-ERK (sc-7383), ERK (sc-94), JNK (sc-7345), p-JNK (sc-6254), p-p38 (sc-166182), and PINK1 (sc-33796) antibodies were acquired from Santa Cruz (Paso Robles, CA, USA). MT1 (NBP1-71113), FKBP4 (NB110-96874), FKBP5 (NBP1-84676), LC3 (NB100-2220) and NIX (NBP1-88558) were purchased from Novus Biologicals (Littleton, CO, USA). TOMM20 (ab56783), p-GR (ab55189), DNMT1 (ab19905), DNMT3a (ab23565), and SP1 (ab227383) antibodies were obtained from Abcam (Cambridge, MA, USA). Cleaved caspase 3 (9661 S), DNMT3b (67259 S), GR (12041 S), 5-methylcytosine (5-mc, 28692 S), p38 (9212 S), IgG rabbit (2729 S), and p-c-MYC ser62 (13748 S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). HSP90 antibody (PA3-013) was obtained from Thermo Fisher (Rockford, IL, USA).