Mouse anti β actin monoclonal igg1 antibody

IPEC-J2 cells were treated with DON in the absence or presence of pretreatment with TLR2 ligands, washed with PBS and lysed in a lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), followed by a quantitation of protein using Micro BCA kit (Thermo, Rockford, USA). For isolation of cytosolic and membrane parts from IPEC-J2 cells, membrane protein extraction kit (Thermo) was used by its instruction. As previously described [17 (link)], the same amount of protein extracts was loaded in 10% Tris–glycine polyacrylamide gels and electrophoresed. Then, the proteins were transferred onto a polyvinylidene difluoride (PVDF) microporous membrane for 2 h at 4 °C and blocked with 5% skim milk in TBS-T (20 mM Tris HCl, 100 mM NaCl, 0.05% Tween 20) for 90 min. The blot was incubated with rabbit anti-claudin-3, -occludin or -zonula occludens (ZO)-1 antibodies (Invitrogen), anti-p-AKT, -p-P70S6K, -Akt, -FAK, and -Bcl-2 antibodies (Cell signaling), or mouse anti-β-actin monoclonal IgG1 antibody (Santa Cruz Biotechnology, Grand Island, USA) overnight. Subsequently, the membrane was washed and incubated with goat anti-rabbit or anti-mouse IgG-HRP (Santa Cruz Biotechnology) for 1 h. The target protein was visualized with enhanced chemiluminescence (ECL) system (GE Healthcare, Waukesha, USA), followed by analysis using ChemiDoc XRS (Bio-rad, Hercules, USA).