Anti rabbit or anti mouse igg conjugated with horseradish peroxidase

Cell lysates were prepared and lysed with 1× RIPA buffer (Millipore, Burlington, MA, USA) with 0.05% protease inhibitor cocktail (Millipore) and 0.05% phosphatase inhibitor cocktail (Millipore). Protein concentrations were measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Cell lysates treated with SDS were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was then blocked with 5% skim milk dissolved in TBS-Tween20 (0.1%, v/v) for 30 min. The membrane was shaken in primary antibodies for overnight in 4 °C cold room. These antibodies were probed with anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase (1:1000, Cell Signaling). The membrane was washed three times with TBS-T after secondary antibody treatment. The bands on the membrane were visualized with Pierce™ ECL western blotting substrate (Thermo Fisher Scientific) and exposed to autoradiography films (Labscientific, Highlands, NJ, USA). The intensity of bands was quantified using ImageJ Software (NIH and Laboratory for Optical and Computational Instrumentation, LOCI, University of Wisconsin, Madison, WI, USA). The ratio of LC3-II/β-actin was normalized to LC3-II/ β-actin in the control (DMSO) group, which was set to 1.

Western blot analysis was performed as previously described [81 (link)]. The following antibodies were used: rabbit anti-TFF3 polyclonal antibody (BioGene GmBH, Berlin) and mouse anti-β-actin (Santa Cruz Biotechnology, USA). The secondary antibody anti-rabbit or anti-mouse IgG conjugated with horseradish peroxidase was from Cell Signaling Technology. Proteins were visualized using horseradish peroxidase conjugated secondary antibody with enhanced chemiluminescence ECL kit (SuperSignal West Pico substrate; Pierce, Rockford, IL).