Irak4

Protein samples were fractionated with 12% SDS‐PAGE (Invitrogen, USA), then transferred into polyvinylidene fluoride membranes (Millipore). The membranes with blotted protein were blocked, followed by probing with TLR4, MyD88, TRIF, NF‐κB, phospho‐NF‐κB, IκB, phospho‐IκB (Cell signal technology), IRAK‐4, phospho‐IRAK‐4 (Santa Cruz) antibodies at 4°C overnight. The membranes were washed and incubated with horseradish peroxidase‐conjugated secondary antibody. Immunoreactive proteins were identified using Super Signal West Pico Chemiluminescence Substrate (Thermo). Densitometric analysis of Western blots was performed with the use of Image J software. GAPDH was used as loading control.

Prepared SDS-PAGE protein samples were subjected to 15% SDS-PAGE gels, and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk-PBS at room temperature for 1 hour and subsequently probed with one of the following antibodies according to experiments: GAPDH (Millipore, Billerica, MA, catalog number MAB374), c-Jun (Millipore, Billerica, MA, catalog number 09–754), NF-κB p65 (Cell Signaling, Danvers, MA, catalog number 3033S), ANT, POLG, TLR9, IRAK4, MYD88, TFAM, and caspase 1 (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc–9299, sc–5933, sc–25468, sc–34470, sc–11356, sc–23588, sc–56036), AP-endonuclease, OGG1, UNG1(Sigma-Aldrich, St. Louis, MO, catalog number A2105, SAB2500714, PRS3865), RAGE (Abcam, Cambridge, MA, catalog number ab3611), or ACS (Novus Biologicals, Littleton, CO, catalog number NBP1-78977). The membranes were then rinsed and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, catalog number 170–6515 and 170–6516). Antibody dilutions and incubation time were according to manufacture’s instructions. Membranes were then rinsed and bound antibodies were detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, catalog number 34077).