Kgaa37

The cells were lysed in equal volumes of ice-cold lysis buffer containing a protease inhibitor cocktail (Pierce Chemical Co.). Cell homogenates were boiled for 5 min in 5× Laemmli sample buffer. Total proteins were extracted with a Whole Protein Extraction kit (KGP250, KeyGEN BioTECH, China). Polyvinylidene difluoride membranes were then blocked in 5% fat-free milk or 5% BSA in TBS containing 0.05% Tween 20. Following overnight incubation at 4 °C with antibodies targeting TLR5 (ab13876, Abcam, UK), MyD88 (ab133739, Abcam, UK), p65 (ab32536, Abcam, UK), phosphorylated p65 (p-p65, ab86299, Abcam, UK) and GAPDH (ab181602, Abcam, UK), the membranes were incubated with HRP-conjugated secondary antibodies (KGAA35, KGAA37, KeyGEN BioTECH, China) at a 1:150,000 dilution for 1 h at room temperature and developed with a SuperSignal chemiluminescent detection system.

Cellular proteins were extracted using a whole-protein extraction kit (KGP250, Keygen). Total protein was measured using a micro bicinchoninic acid protein determination kit (KGA902, Keygen). Thirty micrograms of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Then electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in Tris-buffered saline plus Tween at room temperature for 2 h, then incubated at 4 °C overnight with primary antibody (protein kinase B [Akt], 1:1000 dilution, ab179463, Abcam; p-Akt, 1:5000 dilution, ab81283, Abcam; phosphatidylinositol 3-kinase [PI3K], 1:1000 dilution, ab191606, Abcam; p-PI3K, 1:1000 dilution, ab182651, Abcam) in 1% milk in Tris-buffered saline plus Tween. Finally, they were incubated for 2 h with goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (KGAA37, Keygen). Protein bands were detected using enhanced chemiluminescent reagents (KGP116, Keygen).