Anti flag m2 antibody

Total cell lysates and immunoprecipitates were resolved on 15% tris-glycine SDS-PAGE gel, transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, 1620177) and were detected using an anti-DYKDDDDK Tag (D6W5B) rabbit mAb (binds to same epitope as Sigma’s Anti-FLAG® M2 Antibody) (1:1000 Cell Signaling Technology, 14793S), and 1:5000 dilution anti-rabbit HRP conjugated IgG as the secondary antibody (Cell Signaling Technology, 7074).

After extracting total protein from mouse ileum tissues, immunoprecipitation assay was performed using Pierce Co-Immunoprecipitation (Co-IP) Kit (#26149, Pierce Co-IP Kit, Thermo Fisher Scientific, IL, United States) as the manufacturer instructed. Followed by preclearing lysate with the control agarose resin, tissue proteins were mixed with 1 μg of antibody and then incubated overnight, anti–immunoglobulin G (IgG) antibody as a control. The bound antigens were eluted from the agarose resin using elution buffer. Eluted samples were carried out with sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel electrophoresis. Immunoblotting was carried out as previously described (Chen et al., 2018 (link)). Clean-Blot^TM IP Detection Reagent (horseradish peroxidase) (#21230, Thermo Fisher Scientific) was used as secondary antibody to eliminate IgG bands. Primary antibodies against p16 (#ab211542, Abcam, United States), occludin (#sc-133256, Santa Cruz Biotechnology Inc., United States), and DYKDDDDK Tag (binds the same epitope as Sigma’s Anti-FLAG M2 antibody, #14793, Cell Signaling Technology, United States) were used. The immunoreactive bands were visualized by ECL chemiluminescence (Amersham Pharmacia Biotech, NJ, United States) and analyzed by the Scion image Beta 4.02 (Scion, National Institutes of Health, Bethesda, MD, United States).

Cell lysates were resolved by electrophoresis using 12% SDS-PAGE and transferred to a Hybond P membrane (GE Healthcare). The membrane was probed with an anti-Flag M2 antibody, anti-acetylated-lysine antibody (Cell Signaling Technology, Danvers, MA, USA), or anti-β-actin antibody (G043; Applied Biological Materials Inc., Richmond, BC, Canada). Horseradish peroxidase-labeled anti-rabbit IgG antibody (GE Healthcare) and anti-mouse IgG antibody (GE Healthcare) were used as secondary antibodies. The proteins were detected using ImmunoStar LD (Wako Pure Chemical, Osaka, Japan) and visualized with a LAS-1000 Lumino Image analyzer (Fuji Film, Tokyo, Japan).