Western blotting

Cells were rinsed with cold PBS, and the culture plate was placed on ice. The cell-culture membrane support system was cut from the plastic transwells and placed in lysis buffer with protease inhibitor. Cells were scraped from the membranes, and the suspension was centrifuged at 4°C at 10,000g for 10 minutes. The supernatant was removed, and protein quantification was performed by using a serial bicinchoninic acid assay. The samples in SDS sample buffer were boiled for 10 minutes, separated by using SDS-PAGE gradient gels (Novex Life Technologies, Invitrogen), followed by Western blot analysis. The membranes were blocked with 5% milk and then incubated with primary antibody overnight at 4°C. Primary antibodies included STAT6 (rabbit anti-human, 1:1,000 dilution; Santa Cruz Biotechnology, Dallas, Tex), phosphorylated STAT6 (pSTAT6; rabbit anti-human, 1:1,000 dilution; Santa Cruz Biotechnology), TYK2 (rabbit anti-human, 1:1,000 dilution; Thermo Fisher Scientific, Waltham, Mass), and phosphorylated TYK2 (pTYK; rabbit anti-human, 1:500 dilution; Thermo Fisher Scientific). After serial rinsing with TBS with 20% Tween, the membranes were exposed to secondary antibody (LI-COR Biosciences, Lincoln, Neb) for 1 hour at room temperature. Quantitative expression analysis with 2-color infrared imaging was used to compare protein expression (Odyssey Imager; LI-COR Biosciences).