Laemmli gel loading buffer

Caco-2 monolayers were rinsed twice with ice-cold PBS and lysed using RIPA buffer (Sigma-Aldrich, R0278) containing protease inhibitors (Sigma-Aldrich, 11836170001). The mouse colonic mucosa or human colonic mucosa were washed with ice-cold PBS and snap-frozen in liquid nitrogen soon after harvesting or treatment. The frozen tissues were resuspended in RIPA buffer containing protease inhibitors, minced, and sonicated to prepare tissue lysates. The cell/tissue lysates were centrifuged at 10,000 rpm for 10 min to remove debris, and the protein content of clarified supernatants was quantified using the BCA protein assay kit (Thermo Fisher Scientific, 23225). Laemmli gel loading buffer (Invitrogen, NP007) was added to the lysate, and the samples were boiled at 70°C for 10 min. An equal amount of protein was loaded on an SDS-PAGE gel, separated, and transferred to a nitrocellulose membrane. The membrane was incubated for 1 h in a blocking solution (5% non-fat dry milk in TBS-Tween 20 buffer), followed by incubation with the appropriate primary antibody in the blocking solution. After incubation with the primary antibody, the membrane was washed in TBS-0.1% Tween 20 buffer, then incubated in the appropriate secondary antibody and developed using SuperSignal West Pico PLUS kit (Thermo Fisher Scientific, 34580). The densitometry analysis was performed using ImageJ software.^{73 (link)}