Western Blot Analysis of Inflammatory Signaling

Generally, the lysed protein samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes via electrophoresis, while aliquots of samples were used to determine the protein concentration of each sample using a bicinchoninic acid (BCA) kit (KGPBCA, KeyGEN Biotech, Nanjing, China)^{7 (link),8 (link)}. Subsequently, membranes were incubated in blocking buffer for 2 h and incubated overnight at 4 °C with the following primary antibodies: anti-TLR4 (1:200; SC-10741; Santa Cruz, CA, USA), anti-p-IKKα + β (1:500; ab2064; Abcam, Cambridge, UK), anti-IKKα + β (1:1000; ab178870; Abcam, Cambridge, UK), anti-p-IκBα (1:1000; ab12135; Abcam, Cambridge, UK), anti-IκBα (1:500; ab32518; Abcam, Cambridge, UK), anti-p-NF-κB p65 (1:500; #3033; Cell Signaling Technology, Boston, USA), anti-NF-κB p65 (1:1000; ab16502; Abcam, Cambridge, UK), and anti-HO-1 (1:2000; ab13243; Abcam, Cambridge, UK). GAPDH (1:15,000; ab8245; Abcam, Cambridge, UK) was used as a control on the same membranes. Secondary antibodies were applied, and the bands were detected with West Dura Extended Duration Substrate (Pierce, USA) and X-ray film (Kodak, USA). Then relative density of bands (based on gray value) was analyzed using Bandscan 5.0 software (Glyko, USA) via comparison with GAPDH expression^{7 (link),8 (link)}.