Crosslink magnetic ip co ip kit

Pierce^™ Crosslink Magnetic IP/Co-IP Kit (88805, Thermo Scientific^™, Waltham, Massachusetts, USA) was used in this experiment. In brief, protein A/G magnetic beads were bound and crosslinked by DSS to the first antibody. Then, the cell lysates were incubated with the beads overnight. Finally, the bound antigens were eluted and subsequently analyzed by Western blotting. Anti-EGR1 (ab194357, Abcam, USA) and anti-KAT3B/p300 (ab275378, Abcam, USA) were used as the first antibody.

Cell lysates preparation and immunoprecipitation were conducted using Crosslink Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, Grand Island, NY, USA) according to the manufacturer’s instructions. The cell lysate was incubated with primary antibodies on a rotator overnight at 4 °C, then subjected to western blot for analysis.

Immunoprecipitation assays were performed using the Crosslink Magnetic IP/Co-IP Kit (Thermo Scientific). Cells were solubilized with immunoprecipitation buffer, equal amounts of protein were incubated with specific antibody immobilized onto protein A/G magnetic beads overnight at 4 °C with gentle rotation. Beads were washed extensively with immunoprecipitation lysis buffer, resuspended, and eluted. Protein samples were subjected to SDS-PAGE followed by western blot analysis.
For western blot assay, total protein was extracted from the cultured cells using the total protein extraction kit (KeyGene). The protein content was determined using the bicinchoninic acid protein assay kit (Bio-Rad) with bovine serum albumin as the standard. Proteins (30 μg) were separated by 8–12% SDS–PAGE and transferred onto nitrocellulose filter membranes (Life Science). After blocking in 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20, the membranes were probed with primary antibodies overnight at 4 °C followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at RT. Antigen-antibody complexes were visualized using enhanced chemiluminescence detection kit (Advansta) and image analyzer ImageQuant LAS 500 (GE Healthcare).

Based on the instructions of the Pierce™ Crosslink Magnetic IP/Co-IP Kit (Thermo, 88805), mouse anti-His IgG was incubated with beads at 37 °C for 2 h, and un-crosslinked anti-His was removed using lysis/wash buffer (provided in the kit). His-Eno (His tag was used for the control group) was incubated with beads/anti-His, and un-crosslinked His-Eno or His tag was washed with lysis/wash buffer. A 1 × SDS loading buffer was added to the pellets of anti-His/His-Eno or His tag beads, and then they were boiled at 100 °C for 5 min.

All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. NS1643 was from Alamo Laboratories Inc. (San Antonio, TX, USA). Calpeptin was purchased from Tocris Bioscience (Minneapolis, MN, USA). PTP1B inhibitor was from Cayman Chemical (Ann Arbor, MI, USA). Phospho-Y14, total Caveolin-1, β-catenin, and caspase-3 antibodies were from BD Biosciences (San Jose, CA, USA). Phospho-Y416, Y527, and total Src were from Cell Signaling Technology (Danvers, MA, USA). PTP1B, desmoplakin, desmoglein, and plakophilin monoclonal Abs, normal mouse IgG, and protein A/G agarose beads were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). DAPI and all fluorescently labeled secondary antibodies were purchased from Molecular Probes (ThermoFisher Scientific, Waltham, MA, USA). Active β-catenin monoclonal antibody was purchased from MilliporeSigma (Burlington, MA, USA). HRP-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies were from KPL (Gaithersburg, MD, USA). Lipofectamine 2000, ECL Super Signal kit, and Crosslink magnetic IP/Co-IP kit were from ThermoFisher Scientific (Waltham, MA, USA).

IP assays were conducted with the Crosslink Magnetic IP/Co-IP kit (Thermo, Rockford, IL, USA). The measurements were performed based on the instructions provided by the manufacturer, as previously described [25 (link)].