Claudin 2

Protein concentration was determined by the BCA protein assay [43 (link)]. Membranes were exposed to the following antibodies purchased from Cell Signaling Technology: Occludin (#91131), Claudin-2 (#48120), Cleaved-IL-1β (#63124), IL-18 (#57058), Caspase-1 (#2225), NLRP3 (#15101), ASC/TMS1 (#67824), AIM2(#63660), β-actin (#4970), Malt1(#2494), Bcl10(#4237), Nod1(#3545), Card9 (#12283), Ripk2 (#4142). Anti-ZO-1 antibody purchased from abcam (ab221547), Anti-NLRC4 antibody purchased from ECM Bioscience(#NP5381). Western blotting signals were quantified by a FluorChem densitometer (Alpha Innotech, San Leandro, CA).

All the buffers were supplemented with protease inhibitors (AGScientific) during enterocyte separation. Solubilized proteins were separated by 10% SDS-PAGE. Visualization was by chemiluminescence (SuperSignal, Thermo Fisher, 34580) and digital imaging (Protein Simple, San Jose,). Rabbit anti-mouse ZIP14 antibody was custom-made by Genscript (Piscataway, NJ) (47 (link)). Β-Actin (Cell Signaling, 8457), Claudin 1 (ThermoFisher, 71-7800), Claudin 2 (Cell Signaling, 48120), ZO-1 (ThermoFisher, 61-7300), P-EGFR (Cell Signalling, 3777), P-AKT (Cell Signalling, 4060), AKT (Cell Signalling, 4691), P-STAT3 (Cell Signalling, 9145), STAT3 (Cell Signalling, 30835), Cleaved Caspase 3 (Cell Signalling, 9661), SLC39A4/ZIP4 (Proteintech, 20625-1), SLC39A5/ZIP5 (Novusbio, NBP3-04949). Band intensities were measured by the software ImageJ. Values for protein of interests were normalized to their control (pan-proteins or β-actin). The percentage of changes over control groups was presented.

WB analysis was performed as described previously 15 (link). In brief, after quantification, proteins extracted from cells or colon tissues were separated by SDS-PAGE gels, transferred to nitrocellulose membranes, and blocked with 5% BSA. After incubation with primary antibodies and corresponding HRP-conjugated secondary antibodies, the protein bands were visualized using an ECL substrate (BIO-RAD, 1705061, Hercules, CA, USA). The primary antibodies used for the WB are listed as follows: GAPDH (Cell Signaling Technology, 2118S), P2Y1R (Affinity, DF10258), p-AMPK (Bioworld Technology, BS5003), AMPK (Bioworld Technology, BS1009), ZO-1 (Abcam, ab96587), occludin (Abcam, ab222691), and claudin-2 (Cell Signaling Technology, 48120).