Hrp linked secondary antibody anti rabbit igg or anti mouse igg

Immunoblotting analysis was performed as previously described [19 (link)]. Briefly, protein extracts from jejunal mucosa were separated by 10% SDS-PAGE and then transferred to PVDF membrane. Membranes were blocked with 5% non-fat dry milk in TBST for 1 h and overnight incubated at 4 °C with the primary antibodies against zonula occludens-1 (ZO-1), claudin-1, occludin, and β-actin (Proteintech, Chicago, IL, USA), followed by incubation with HRP-linked secondary antibody anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, Danvers, USA) in TBST for 1 h at room temperature. Finally, membranes were visualized using Bio-Rad ChemiDoc™ imaging system (Bio-Rad). Band density of target protein was quantified after normalization to β-actin.

Total protein was extracted by lysing samples in RIPA buffer with 1% PMSF, and the protein levels were quantified by a protein quantitative analysis kit (KeyGEN, China). Cell lysates were diluted at a ratio of 1:5 with protein loading buffer (6×) and heated at 100°C for 5 min. Thirty micrograms of each protein sample was separated on 10% SDS-PAGE gels at 100 V for 2 h and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, United States) for 120 min at 100 mA. After blocking in 5% nonfat dried milk in TBST for at least 1 h, the membranes were incubated with the primary antibodies listed under Reagents in TBST overnight at 4°C. After washing three times with TBST, the bands were then incubated with HRP-linked secondary antibody anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology). The results were visualized via the SuperSignal West Pico Chemiluminescent Substrate kit (Thermo, United States). Densitometry of the immunoreactive bands was performed with Tanon Image software. GAPDH was regarded as an internal control.