Odyssey clx fluorescence scanning system

Total colon tissues and BMSCs cells were extracted by RIPA lysis buffer with 0.1% PMSF (Beyotime, China). Protein quantification was performed with the BCA assay kit (Beyotime). The homogenized protein samples were fractionated by 4%–20% pre‐cast gel (GenScript) at 120 V for 1.5 h, and transferred onto nitrocellulose filter (NC) membranes (Bio‐Rad Laboratories,USA) at 110–120 V for 1 h. The membranes were blocked in blocking buffer (Beyotime, China) for 15–30 min at room temperature, followed by an overnight incubation at 4°C with primary antibodies (Anti‐Occludin, #91131, 1:1000; Anti‐E‐cadherin, #14472, 1:1000; Anti‐LC3B, ab192890, 1:1000; Anti‐Caspase‐3, ab32351, 1:1000), and respective fluorescent secondary antibodies (Goat anti‐Mouse IgG H&L IRDye 680RD, ab216776, 1:5000; Goat anti‐Rabbit IgG H&L IRDye 800CW, ab216773, 1:5000) at 37°C for 1–2 h. Finally, the immunoreactive bands were visualized using Odyssey Clx fluorescence scanning system (LI‐COR Biosciences, USA). Equal protein loading was normalized with Anti‐β‐actin antibody.

Total proteins from brain tissues and PC-12 cells were extracted by RIPA lysis buffer with 0.1% PMSF (P0013C, Beyotime, China). BCA protein assay kit (P0012, Beyotime, China) detected total protein concentration. The homogenized protein samples were fractionated by 4-20% precast gel (M42015C/M42010C GenScript, China) at 120 V for 1.5 h and transferred onto nitrocellulose filter (NC) membranes (Bio-Rad Laboratories, Hercules, USA) at 110–120 V for 1 h. The membranes were blocked in blocking buffer (Beyotime, Shanghai, China) for 15-30 min at room temperature, followed by an overnight incubation at 4°C with primary antibodies (Anti-LC3B, ab192890, 1 : 1000; Anti-SQSTM1/P62, #5114, 1 : 1000; Anti-Atg4B, ab154843, 1 : 1000; Anti-Bcl-2, ab59348, 1 : 1000; Anti-Beclin 1, ab207612, 1 : 1000; Anti-AMPKα(D5A2), #5831, 1 : 1000; Anti-p-AMPKα(Thr172)(40H9), #2535, 1 : 1000; Anti-ZO-1, ab96587, 1 : 500; Anti-β-actin, #3700, 1 : 1000) and respective fluorescent secondary antibodies (Goat anti-Mouse IgG H&L IRDye® 680RD, ab216776, 1 : 5000; Goat anti-Rabbit IgG H&L IRDye® 800CW, ab216773, 1 : 5000) at 37°C for 1-2 h. Finally, the immunoreactive bands were visualized using Odyssey CLx fluorescence scanning system (LI-COR Biosciences, USA). Equal protein loading was normalized with Anti-β-actin antibody.