Acetylated lysine ac k

Protein samples (16–20 μg) were loaded on 7.5–15% poly-acrylamide gels, separated by sodium dodecyl sulphate/poly-acrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using the Semi-Dry Trans-Blot Cell (Bio-Rad Laboratories, Hercules, CA, USA). After the transfer, the membranes were blocked for 1-h with 5% (w/v) skim milk or bovine serum albumin powder in Tris-buffered saline with 0.1% Tween-20 at room temperature. Blots were incubated with primary antibodies overnight at 4°C. Primary antibodies against MyD88, TIR-domain-containing adapter-inducing interferon-β (TRIF), nuclear factor kappa B (NF-κB), inhibitor of kappa B (IκB)-α, acetylated-lysine (Ac-K; Cell Signaling Technology, Berverly, MA, USA), TLR4 and TLR2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used. On the following day, the blots were incubated with appropriate secondary antibodies for 1-h at room temperature. Bands were detected using an ECL detection system (iNtRON Biotechnology) according to the manufacturer’s instructions. The intensities of immunoreactive bands were evaluated using Total-Lab TL120 software (Nonlinear Dynamics, New-castle, UK). Signals were standardized to that of β-actin (Sigma-Aldrich) and lamin B1 (Abcam, Cambridge, MA, USA) for whole/cytosolic lysate and nuclear fraction, respectively.