Mouse anti beclin 1

A375 cells were treated with TanII A (1, 2, or 4 μg/mL) for 48 h in 6 well plate (5 × 10⁵ cell/2 ml) at 37 °C and 5% CO2. The cells with 0.1% DMSO was used as negative control. At the time of harvest, cells were washed with PBS, and whole cell protein samples were extracted with radioimmunoprecipitation assay buffer (0.5% Nonidet p-40, 10 mM Tris, pH 7.4, 150 mM NaCl, 1% SDS) with a protease inhibitor cocktail (Sigma Life Science and Biochemicals, St. Louis, MO). Protein (70 μg/well) was separated by SDS-PAGE and transferred to PVDF membranes. The primary antibodies used for the Western blots included mouse anti-Beclin-1, mouse anti-microscopic light chain I protein 3 (the microtubule-associated protein light chain3, LC3) -II, and mouse anti-β-actin (Santa Cruz Biotechnology, TX, USA); mouse anti-phosphatidylinositol 3-kinase (PI3K), mouse anti- phosphorylated (p) - protein kinase B (P-Akt), p- mammalian target of rapamycin (P-mTOR), p-p70S6K1 (Cell Signaling Co, MA, USA). The secondary antibodies included HRP-conjugated ECL sheep anti-mouse IgG (GE Amersham Biosciences, USA). The ECL Western Blotting Detection Kit (GE Amersham Biosciences, USA) was used to detect Chemiluminescent signals in the X-ray film. The GAPDH was used as internal protein level control. Each experiment was repeated three times.

The extraction of cellular proteins and their detection via western blotting were performed as previously described [21 (link), 24 (link)]. Western blotting was performed using the following primary antibodies: mouse anti-AR, mouse anti-beclin1, goat anti-LC3β, mouse anti-LAMP1, and rabbit anti-IKKγ antibodies (1:500, Santa Cruz Biotechnology); mouse anti-TLR4, rabbit anti-iNOS, rabbit anti-IKKα, and rabbit anti-4-HNE (1:1000, Abcam); rabbit anti-IKKβ and rabbit anti-phospho-IκBα antibodies (1:1000, Epitomics); rabbit anti-phospho-IKKα/β, rabbit anti-IκBα, rabbit anti-p65, and rabbit anti-phospho-p65 antibodies (1:1000, Cell Signaling Technology, Danvers, MA, USA); and mouse anti-β-actin antibodies (1:8000, Sigma-Aldrich). The blots were then incubated with their respective secondary antibodies: horseradish peroxidase-conjugated goat anti-rabbit IgG, goat anti-mouse IgG (both 1:8000, Abcam), and donkey anti-goat IgG (1:5000, Santa Cruz Biotechnology). β-actin was used as the loading control. The immunoreactive bands were scanned using the Bio-Rad ChemiDoc™ XRS⁺ imager with Image Lab™ Software (Bio-Rad Laboratories, CA, USA). Band intensity was quantified using Quantity One software (Bio-Rad Laboratories).