Imagequant las 4000 mini biomolecular imager

Cells with 0 or 5 mM nicaraven treatment were harvested, washed with PBS, and suspended in Laemmli's buffer. For the extraction of nuclear content and cytoplasmic fractions, Nuclear Extraction Kit (Abcam, UK) was used. Cell lysates were heated at 95˚C for 5 min, then loaded on SDS-PAGE gel and transferred to PVDF membrane (Bio-Rad Laboratories, US). Membranes were probed with antibodies against PARP-1, PARG, AIF, caspase-3, caspase-7, caspase-9, β-Actin, α-Tubulin (1:1000; Cell Signaling Technology, US), PAR (1:1000; Trevigen, US), Bcl-2 (1:1000; Bethyl laboratory, US), and Lamin B1 (1:1000; Abcam, UK) at 4°C overnight or 25°C for 1 h. Followed by the appropriate secondary antibodies for 1 h at 25°C, the expression was visualized using an ECL detection kit (SuperSignal West Femto, ThermoFisher Scientific, US). The blots were detected using ImageQuant LAS 4000 Mini biomolecular imager (Cytiva, US) and analyzed by ImageJ 2.1.0 software (National Institutes of Health, US).

Western blotting was performed as previously described (21 (link)). Briefly, cells were lysed in Laemmli's buffer. Total proteins were separated using SDS-PAGE and were then transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking, the membranes were incubated with primary antibodies against rabbit phosphorylated (p)CREB (Ser133; 1:1,000, cat. no. ab32096; Abcam), rabbit PARP-1 (1:1,000 cat. no. 9542; Cell Signaling Technology, Inc.) and mouse α-tubulin (1:1,000, cat. no. 3873; Cell Signaling Technology, Inc.) which was followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies against rabbit (1:2,000, cat. no. p0448; Dako Agilent Technologies) and mouse (1:2,000, cat. no. p0260; Dako Agilent Technologies). The expression was visualized using an ECL detection kit (cat. no. RPN2106; Cytiva). Images were acquired using ImageQuant LAS 4000 Mini biomolecular imager (Cytiva). Semi-quantification on the relative expression of proteins was performed using ImageJ 2.1.0 software (National Institutes of Health).