Irdye800cw conjugated secondary antibody

Western blotting analysis for CE7R expression was performed using an anti-human CD3ζ chain (cytoplasmic tail)-specific monoclonal antibody 8D3 (BD Pharmingen), followed by goat anti-mouse IgM (mu chain) IRDye^® 800CW conjugated secondary antibody (Rockland Immunochemicals Inc), and imaging with the Odyssey Infrared Imaging System (LI-COR).

Total protein was isolated from murine BMDMs and colonic tissues using a standard extraction reagent supplemented with protease inhibitor (Kangchen; Shanghai, China). The concentration of extracted protein was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were then separated using SDS-PAGE and electro-transferred to nitrocellulose membranes as previous described [50 (link)]. Immunoblotting was performed using a Beclin-1 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), an LC3 antibody (1:500; Novus Biologicals, Littleton, CO, USA), and a p62 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA). The membranes were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at 25 °C. Finally, images of the blots were obtained using an Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA).

Proteins were extracted from the colonic tissues or murine BMDM using a standard extraction reagent supplemented with the protease inhibitor (KANGCHEN; Shanghai, China). The protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Proteins were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro-transferred to nitrocellulose membranes as described previously^{68 (link)}, and incubated with a primary antibody overnight at 4 °C. The samples were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at 25 °C. The image was acquired with the Odyssey infrared imaging system (Li-Cor Biosciences, Lincoln, NE, USA). All of the immunoblotting experiments were repeated at least five times. The following primary antibodies were used: Beclin-1 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), light chain 3 (LC3) antibody (1:500; Novus Biologicals, Littleton, CO, USA), and p62 antibody (1:500; Cell Signaling Technology, Danvers, MA, USA), CD68 antibody (1:300; Proteintech Group Inc., Chicago, IL, USA), and CD206 antibody (1:500; Proteintech Group Inc., Chicago, IL, USA).

Proteins were extracted from muscle tissue using a standard extraction reagent supplemented with protease inhibitors (Kangchen, Shanghai, China). Protein concentration was determined using a bicinchoninic acid method (Beyotime). The proteins were separated using SDS-PAGE and electrotransferred to nitrocellulose membranes and then incubated with a primary antibody: p-JAK2 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), JAK2 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), p-STAT3 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), STAT3 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500) for 8–12 h at 4 °C. Samples were then incubated with an IRDye800CW-conjugated secondary antibody (Rockland, Gilbertsville, PA, USA) for 1 h at 25 °C. The image was acquired with an Odyssey infrared imaging system (Li-Cor Bioscience, Lincoln, NE, USA). All immunoblotting experiments were repeated at least three times36 (link)37 (link).