Protein buffer

Western blot was performed to detect protein expression as previously described (Yuan et al. 2012 (link)). Briefly, total proteins from primary rat cortical neurons were isolated using the protein buffer (Beyotime, Shanghai, China). Proteins were then separated by 10% SDS-PAGE and transferred onto PVDF membranes. After being blocked with 5% fat-free milk, the membranes were incubated at 4 °C overnight with primary antibodies against p62 (1:1,000, Abcam, Cambridge, MA, USA), Beclin-1 (1:1,000, Abcam), microtubule-associated protein 1 light chain 3 (LC3-I and LC3-II, both from Anti-LC3B antibodies, 1:1,000, Sigma-Aldrich), CaMKKβ (1:1,000, Abcam), AMPK (1:1,000, Cell Signaling Technology Inc., Danvers, MA, USA), phospho (p)-AMPK (1:1,000, Cell Signaling Technology Inc.), mTOR (1:1,000, Cell Signaling Technology Inc.), and p-mTOR (1:1,000, Cell Signaling Technology Inc.). Subsequently, membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-secondary antibodies in TBST. The protein was visualized using the ECL detection system (Beyotime, Shanghai, China). The band intensity was quantified with Image-Pro Plus 6.0 software.

Total proteins from IEL cells were lysed using a protein buffer (Beyotime, China). Protein concentration was determined using the BCA protein quantitative kits (Sigma, USA). The extracted protein samples were separated by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were then blocked with 7.5% fat-free milk overnight and incubated with the primary antibodies against NOD2, Beclin 1, Atg16, and GAPDH (at a 1:1,500 dilution; Abcam, USA) at room temperature for 1 h. After washing with tris-buffered saline with Tween-20, the membranes were cultured along with secondary antibodies at room temperature for 1 h (1:5,000; Abcam). Finally, the protein bands were analyzed using the enhanced chemiluminescence detection kits (BestBio, China) according to the manufacturer’s instructions.