Horseradish peroxidase conjugated secondary igg

Cell lysates were prepared and analyzed as previously described. Blots were probed with antibody against Parkin, Thap11 or β-actin (1:500, Santa Cruz Biotechnology, Dallas, Texas, USA) following by horseradish peroxidase-conjugated secondary IgG (1:3000; EMD Millipore, Burlington, Massachusetts, USA).
Densitometry was performed to quantify the protein expression levels using the National Institutes of Health’s ImageJ software.

Cell lysates were prepared and analyzed as previously described^{46 (link)}. Blots were probed with antibody against LMP7 (1:1000, Proteintech), A20 (1:1000, Novus) or β-actin (1:500, Santa Cruz Biotechnology) followed by horseradish peroxidase-conjugated secondary IgG (1:3000; EMD Millipore, Burlington, Massachusetts, USA). Densitometry was performed using the National Institutes of Health’s ImageJ software.

A549 and MRC-5 cells were treated with Acr as indicated, then cell lysates were prepared and analyzed as previously described [59 (link)]. Briefly, blots were probed with a monoclonal antibody against ATG7 (1:1000, Cell signaling, #2631), caspase-3 (1:1000, Cell signaling, # 9662), caspase-9 (1:1000, Cell signaling, # 9502), Drp1 (1:1000, BD, # 611112), LC3 (1:1000, Cell signaling, # 2775), OPA1 (1:1000, BD, # 611112), PARP-1 (1:1000, Cell signaling, # 9542), Pink1 (1:1000, BD, # BC100-494), p-Drp1 (Ser616, 1:1000, Cell signaling, # 3455) and β-actin antibody (1:5,000; Millipore [clone C4]) at 4 °C for overnight following by horseradish peroxidase-conjugated secondary IgG (1:3,000; Millipore) for 1 h at room temperature. The immunoreaction was visualized using Enhanced Chemiluminescence (ECL) (Millipore Corporation, Billerica, MA).

HeLa or A549 cells were treated with Acr (0–100 μM, 3 h), cell lysates were prepared and analyzed as described previously [4 (link)]. Briefly, blots were probed with a monoclonal antibody against MDM2 (1:1000, Abcam, ab178938), p-MDM2 (Ser166, 1:1000, Cell signaling, #3521), E2F-1 (1:1000, Cell signaling, #3742), p-p53 (Ser15, 1:1000, Cell signaling, #9284), p53 (1:1000, Calbiochem) and RPL11 (3A4A7, Thermo Scientific) at 4°C for overnight following by horseradish peroxidase-conjugated secondary IgG (1:3,000; Millipore) for 1 h at room temperature. The immunoreaction was visualized using Enhanced Chemiluminescence (ECL) (Millipore Corporation, Billerica, MA). The bound primary and secondary antibodies were stripped by incubating the membrane in stripping buffer (100 mM 2-mercaptoethanol, 2% SDS) for 30 min at room temperature. The membrane was then re-probed with β-actin antibody (1:5,000; Millipore [clone C4]).