Pierce ecl plus western blot substrate

Cells were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% Na-deoxycholate, and a protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany). For protein isolation from tissue samples, approximately 30 mg tissue was chopped and homogenized on ice. Protein concentrations were determined by the Pierce Protein BCA Assay Kit (Thermo Fisher Scientific Inc., Waltham, United States of America). Primary antibodies used to detect the individual target proteins and corresponding secondary antibodies are shown in Supplementary Table S3. Pierce ECL Plus Western Blot substrate (Thermo Fisher Scientific Inc., Waltham, United States of America) and the FluorChem Q detection system (Bio-Techne Corp., Minneapolis, United States of America) were used for visualization of protein signals. All Western blots shown are representative for at least two independent experiments.

EAhy926 cells were incubated with serum-free DMEM media (blank), C, R, or CR at 50 μg/mL in T75 cell flasks for 24 hours. The cells were then lysed with RIPA lysis buffer (Thermo Fisher Scientific, Australia) and the protein was collected and quantified using BCA quantification kit (Thermo Fisher Scientific, Australia). Equal amounts of proteins were then separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked by 5% milk and then incubated overnight at 4°C with rabbit polyclonal antibodies against heme oxygenase-1 (HO-1, 1 : 1000, Cell Signaling Technology, USA) and beta-actin (1 : 1000, Cell Signaling Technology, USA), followed by anti-rabbit horseradish peroxidase-conjugated secondary antibodies. Then, the membranes were exposed to Pierce ECL Plus western blot substrate (Thermo Fisher Scientific, Australia). The band intensities of the membranes were quantified by ImageJ, and the control for equivalent protein loading was assessed by anti-β-actin antibody.

Cells were collected in resuspension buffer (100 mM NaCl, 15 mM MgCl₂, 100 mM Tris, pH 7.5) and incubated on ice for 10 min while vortexing regularly. Samples were homogenised by flushing the cells 5 times through a G25 needle. Subsequently, nuclear (NER) and mitochondrial (MER) protein fractions were collected using differential centrifugation^{56 (link)}. Protein quantification was performed with the DC BioRad Protein Assay (BioRad). 50 µg protein was loaded on a 12% SDS-PAGE gel for the detection of the mitochondria-targeting construct (containing a HAtag). Blots were blocked for 1 h with 5% skimmed milk in TBS. For detection, primary antibodies were incubated O/N at 4 °C, whereas secondary antibodies were incubated for 1 h at RT. The following antibodies were used: 1:1000 mouse anti-HAtag (HA.11, Biolegend), 1:1000 rabbit anti-VDAC1/Porin (Ab34726, Abcam), 1:1000 mouse anti-lamin B1 (clone L5, Invitrogen), and 1:1000 horseradish peroxidase-conjugated rabbit anti-mouse (P0260, Dako) and swine anti-rabbit (P0217, Dako). Western blot signal was generated with Pierce ECL Plus Western blot substrate (Thermo Scientific) and detected with the Biorad ChemiDoc MP imaging system (Biorad).