Bcl2 associated x (bax)

To determine the impact of knocking down Nrf2 on its target genes
HO1 and NQO1 protein expression, cell lysates were collected and analyzed
on 10% NuPAGE gels as detailed in Madhunapantula et al.^{58 (link)} Total protein quantity was determined using
the BCA assay.^{59 (link)} About 50 μg of total
protein/well was loaded and analyzed as described in Madhunapantula
et al.^{58 (link)} Expression of Nrf2, HO1, NQO1
and p53, Bax, Cyclin-D1, and P27 was measured by detecting the respective
proteins using primary antibodies recognizing Nrf2 (cat no. 12721),
NQO1 (cat no. 62262), HO1 (cat no. 5853), p53 (cat no. 9282S), Bax
(cat no. D2E11), Cyclin-D1 (cat no. SC718), and P27 (cat no. SC 393380)
obtained from Cell Signaling Technologies, Danvers, MA. Enolase (cat
no. SC-7455) and secondary antibodies (antirabbit and antigoat) conjugated
with horseradish peroxidase (HRP; rabbit cat no. SC2357 and goat cat
no. SC2020) were from Santacruz Biotechnology, Dallas, TX. The proteins
were detected using ECL (Western Bright ECL cat no. K-12045-D20),
Advansta Corporation, San Jose, CA.

The total protein expression of cleaved caspase-3 (C-caspase-3; 1:1,000; cat. no. 9661s; Cell Signaling Technology, Inc.), Bax (1:1,000; cat. no. 2772s; Cell Signaling Technology, Inc.) and B-cell lymphoma 2 (Bcl-2; 1:1,000; cat.no. 59348; Abcam) and GAPDH (1:1,000; cat. no. AF1186; Beyotime Institute of Biotechnology) in tumor tissues was analyzed by western blotting. Western blotting was performed as previously described (23 (link)). Briefly, after quantification with a BCA kit (cat. no. P0012S, Beyotime Institute of Biotechnology), 20 µg of protein samples were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The nitrocellulose membrane was then blocked with 5% nonfat milk for 1 h at room temperature. After washing, the nitrocellulose membrane was incubated with C-caspase-3 (1:1,000; cat. no. 9661s; Cell Signaling Technology), Bax (1:1,000; cat. no. 2772s; Cell Signaling Technology), Bcl-2 (1:1,000; cat. no. 59348, Abcam, Cambridge, UK) and GAPDH antibody (1:1,000; cat. no. AF1186; Beyotime Institute of Biotechnology, China) overnight at 4˚C. After incubation with the secondary antibody (1:2,000; cat. no. A0181; Beyotime Institute of Biotechnology) for 2 h at room temperature, the membranes were then visualized using an ECL kit (Tanon Science and Technology Co., Ltd.) on the Tanon Imaging System.

Protein levels in the extracts of SH-SY5Y cells or the hippocampal tissues were analyzed using western blot as previously described [5] (link) with primary antibodies against Thr205-phosphorylated tau-, Tyr307-phosphorylated protein phosphatase 2 A (PP2A)-, total PP2A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Thr181-phosphorylated tau-, total tau-, Thr202/Tyr204-phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2)-, Thr180/Tyr182-phosphorylated p38 mitogen activated protein kinase (p38 MAPK)-, Thr183/Tyr185-phosphorylated c-Jun N-terminal kinase (JNK)-, β-actin-, total ERK1/2-, total p38-, total JNK-, hypoxia-inducible factor-1α (HIF-1α)-, B-cell lymphoma-2 (Bcl-2)-, Bcl2-associated X (Bax)-, cleaved caspase-3-specific antibodies (Cell Signaling Technology, Beverly, MA, USA). Immunoreactive bands were detected with the appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and immunological complexes were visualized by enhanced chemiluminescence reagents (Millipore, Billerica, MA, USA). Western blot signals were analyzed by ImageJ software.