Uncleaved caspase 3

Protein extracts and diluted in sodium dodecyl sulphate loading buffer were electrophoresed on 10% or 15% sodium dodecyl sulfate‐polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Bio‐Rad Lab, Hercules, CA, United States). The membranes were blocked with 5% non‐fat milk for 2 hours, and then incubated overnight at 4°C with Nrf2 (1:1000, Abcam, Cambridge, MA, USA), Bcl‐2 (1:200, Santa Cruz, CA, USA), Cleaved caspase‐3 (1:1000, Cell Signaling, Danvers, MA, USA), Bax (1:200, Santa Cruz, CA, USA), histone H3 (1:1000, Cell Signaling Technology, Beverly, MA, USA), UnCleaved caspase‐3 (1:400, Cell Signaling, Danvers, MA, USA), HO‐1 (1:200, Santa Cruz Biotecnology), NQO1 (1:1000, Abcam, Cambridge, MA, USA), β‐actin (1:5000, Bioworld Technology, MN, USA). After being washed with TBST for three times (15 minutes each), the membranes were subsequently incubated in the secondary antibody (1:1000, Bioworld Technology, MN, USA) at room temperature for 2 hours. The protein bands were exposed to Tanon‐5200 Chemiluminescent Imaging System and strip grey levels were quantified by software (Bio‐Rad Laboratories, Inc).

Protein was extracted, diluted in sodium dodecyl sulfate loading buffer, electrophoresed on 10 or 15% sodium dodecyl sulfate‐polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (Bio‐Rad Lab, Hercules, CA). The membranes were blocked with 5% nonfat milk for 2 h, then incubated overnight at 4°C with Nrf2 (1:1000, Abcam, Cambridge, MA), NF‐κB (Cell Signaling Technology, Beverly, MA), Bcl‐2 (1:200, Santa Cruz, CA), Cleaved caspase‐3 (1:1000, Cell Signaling, Danvers, MA), Bax (1:200, Santa Cruz, CA), NF‐κB (1:1000, Cell Signaling, Danvers, MA), unCleaved caspase‐3 (1:400, Cell Signaling, Danvers, MA), HO‐1 (1:200, Santa Cruz Biotechnology), NQO‐1 (1:1000, Abcam, Cambridge, MA) and β‐actin (1:5000, Bioworld Technology, MN). After washing three times with TBST (15 min each), the membranes were incubated with the secondary antibody (1:1000, Bioworld Technology, MN) at room temperature for 2 h. The protein bands were exposed to the Tanon‐5200 Chemiluminescent Imaging System and quantified using the associated software (Bio‐Rad Laboratories, Inc.).