Horseradish peroxidase labeled anti rabbit igg

Kidney cortex and colon tissues were lyzed in 1 ml radioimmunoprecipitation assay lysis buffer containing 1 mM phenylmethyl sulfonyl fluoride and 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific). A bicinchoninic acid protein detection kit was used to detect protein concentrations. Protein samples (50 μg) were boiled with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, then electrophoresed on a 10% polyacrylamide gel under denaturing conditions and wet-transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, United States). Membranes were exposed to blocking buffer for 2 h and hybridized with primary antibody against ZO-1 (1:1,000; Abcam Cat# ab96587, Cambridge, England), Occludin (1:1,000; Abcam Cat# ab168986, Cambridge, England), Claudin-1 (1:500; Abcam Cat# ab15098, Cambridge, England), IL-22 (1:1,000, Abcam Cat# ab5984, Cambridge, England) or ß-actin (1:2,000; Cell Signaling Technology Cat# 4970, Boston, United States) overnight at 4°C, followed by horseradish peroxidase-labeled anti-rabbit IgG (1:3,000; Cell Signaling Technology Cat# 7074, Boston, United States) at room temperature. Membranes were washed and then visualized using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, United States). Image Lab System (Bio-Rad) was used to captured and analyzed the signals.

Kidney tissues were lysed in 1 mL radioimmunoprecipitation assay lysis buffer containing 1 mM phenylmethyl sulfonyl fluoride and 1% phosphatase inhibitor cocktail (Thermo Fisher Scientific). A bicinchoninic acid protein detection kit was used to detect protein concentrations. Protein samples (50 μg) were boiled with sodium dodecyl sulfate polyacrylamide gel electrophoresis loading buffer, then electrophoresed on a 10% polyacrylamide gel under denaturing conditions and wet-transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). Membranes were exposed to blocking buffer for 2 h and hybridized with primary antibody against aryl hydrocarbon receptor (AhR) (1:500; Abcam, Cat# ab84833 Cambridge, UK), CYP1A1 (1:400; Santa Cruz Biotechnology, Cat# sc-25304, CA, USA), CyP1B1 (1:6000; Abcam, Cat# ab185954, Cambridge, UK), or β-actin (1:2000; Cell Signaling Technologies, Cat# 4970S, Boston, USA) overnight at 4 °C, followed by horseradish peroxidase-labeled anti-rabbit IgG (1:3000, Cell Signaling Technologies, Cat# 7074S, Boston, USA) or anti-mouse IgG (1:3000, Cell Signaling Technologies, Cat# 7076S, Boston, USA) at room temperature. Membranes were washed and then visualized using an enhanced chemiluminescence detection system (Bio-Rad, Hercules, CA, USA). The signals were captured and analyzed using Image Lab System (Bio-Rad).