Polyclonal anti β actin antibody

Formononetin, actinomycin D (Act D), cycloheximide (CHX), Ponceau S solution, resveratrol, sirtinol, MTT, LPS (Escherichia coli 0111:B4), curcumin, genistein, and an anti-β-actin polyclonal antibody were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). GSK0660 and the luciferase assay system were purchased from Tocris Bioscience (Bristol, UK) and Promega (Madison, WI, USA), respectively. Monoclonal antibodies specific for HMGB1 and PPARδ were supplied by Epitomics (Burlingame, CA, USA). Monoclonal antibodies specific for acetyl-lysine, lamin B, and α-tubulin as well as a polyclonal antibody specific for SIRT1 were supplied by Santa Cruz Biotechnology (Dallas, TX, USA).

Thirty µg of extracted total proteins were separated in SDS-PAGE and transferred to nitrocellulose membranes. After being blocked with TBS-T (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 0.1% Tween-20) supplemented with 5% BSA (Bovine Serum Albumin), membranes were blotted with anti-HER2 monoclonal antibody (clone D8F12) (Cell Signaling Technology, Danvers, MA, USA). Moreover, membranes were blotted with an anti-β-actin polyclonal antibody (Sigma–Aldrich, St. Louis, MO, USA) used as a control for equal loading. Images were acquired and digitally scored with a densitometer image analyzer (Quantity one software, Bio-Rad, Hercules, CA, USA). Four independent samples were analyzed for each group of non-operated animals. Densitometric analysis was performed normalizing protein expression to β-actin expression. Data were presented as the mean ± SEM (Standard Error of the Mean). Data on dysplastic tissue represented the mean of n = 6 and n = 4 independent samples from WT and MKR mice, respectively. Data on ESCC tissue represented the mean of n = 5 and n = 6 independent samples from WT and MKR mice, respectively. Data on EASC tissue represented the mean of six independent samples for each group of animals. Relative protein expression was represented as n-fold of protein amount detected on WT dysplastic tissue which was set equal to 1 arbitrary unit.

Western blotting of brain tissues were performed as previously described [18 (link)]. The cerebral cortexes of mice were homogenized in lysis buffer (50 mM HEPES, pH7.2, 150 mM NaCl, 5 mM MgCl₂) containing cOmplete™ EDTA-free protease inhibitor (Roche Diagnostics, Basel, Switzerland)) and centrifuged at 1,000 g for 5 min. The tissue lysates (20 μg of protein) were separated on SDS-PAGE (10% acrylamide gel). The separated proteins were transferred to a polyvinylidene difluoride membrane (Pall Life Sciences, Port Washington, NY). The membrane was blocked with 5% skim milk and incubated with an anti-ERK1/2 antibody (1:1000 dilution, BRID: AB_330744, Cell Signaling Technology, Danvers, MA), an anti-phospho-ERK1/2 antibody (1:1000 dilution, BRID: AB_331646, Cell Signaling Technology) or an anti-β-actin polyclonal antibody (1:1000 dilution, BRID: AB_476693, Sigma-Aldrich, St Louis, MO) overnight at 4 °C. The immunoreactive bands were visualized using a peroxidase-conjugated anti-rabbit IgG antibody (1:10000 dilution, BRID: AB_2337943, Jackson ImmunoResearch Laboratories, West Grove, PA) and the ECL Western Blotting Detection System (GE Healthcare Bio-Sciences, Piscataway, NJ).