Anti β tubulin

7,8-dihydroxyflavone (7,8-DHF) was purchased from Tokyo Chemical Industry (TCI; Tokyo, Japan) and was dissolved in dimethyl sulfoxide. Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies from Sigma-Aldrich (St. Louis, MO, USA) were used: anti-p53 (C-11), anti-p21 (F-5), anti-cyclin B (GNS1), anti-CDK1 (AN21.2), anti-Mcl-1 (22), anti-survivin (D-8), anti-Bax (P-19), anti-caspase 3 (L-18), anti-caspase 9 (96.1.23), anti-cleaved-caspase 3 (h176), anti-cytochrome c (A-8), anti-GM3 synthase (T-19), anti-GD3 synthase (B-11), anti-Bcl-xL (H-5), and anti-β-actin (C-4) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The other antibodies (anti-Bid, anti-PARP, anti-MTCO1, and anti-tubulin β) were purchased from Cell Signaling Technology (Danvers, MA, USA) and Novus Biologicals, LLC (Centennial, CO, USA).

Quercetin was acquired from Tokyo Chemical Industry (Tokyo, Japan) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) to prepare the stock solution. All remaining reagents were procured from Sigma-Aldrich (St. Louis, MO, USA). The following antibodies obtained from Santa Cruz (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were utilized: anti-p53 (C-11), anti-cyclin D (M-20), anti-cyclin E (M-20), anti-cdk4 (C-22), anti-cdk6 (DSC-90), anti-Bax (P-19), anti-Bcl-xL (H-5), anti-caspase 3 (L-18), anti-cleaved caspase 3 p11 (h176), anti-caspase 8 p18 (D-8), anti-caspase 9 (96.1.23), anti-cleaved caspase 9 p10 (h331), anti-cytochrome c (A-8), anti-GM3 synthase (T-19), anti-GD3 synthase (B-11), and anti-β-actin (C-4). Additional antibodies (anti-Bid, anti-PARP, anti-MTCO1, and anti-tubulin β) were sourced from Cell Signaling Technology (Danvers, MA, USA) and Novus Biologicals (Novous Biologicals, LLC, Centennial, CO, USA).

The Western blot was performed as previously described [25 (link)]. Liver protein extracts were obtained by lyses homogenized tissue in Ripa buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein samples (40 μg) were resolved on 10% or 12% Tris-HCl polyacrylamide gels and subsequently transferred to a nitrocellulose membrane. Membranes were probed with commercially available rabbit polyclonal anti-ApoE (1:500 dilution), anti-GRP-78 (1:500) (Abcam, Cambridge, MA, USA), anti-β-tubulin (1:200) (Cell Signaling, Danvers, MA, USA), anti- ERP29 and anti-α-tubulin (1:2000), anti-SREBP -1c and anti-SOD2 (1:500) antibodies (Abcam, Cambridge, MA, USA), followed by HRP-conjugated anti-rabbit antibody (1:10000) and ECL Plus detection reagents (GE Biosciences, Piscataway, NJ, USA). Relative ApoE, GRP-78, β-tubulin, α-tubulin, ERP29, SREBP and SOD2 band densities were determined by densitometrical analysis using the Image Studio Lite software from LI-COR Corporate Offices-US (Lincoln, Nebraska USA). In all instances, density values of bands were corrected by subtraction of the background values. The results were expressed as the ratio of ApoE, GRP-78, ERP29 and SOD2 to that of β-tubulin and SREBP to that of α-tubulin our β-tubulin.