Claudin 1

Cells were lysed with lysis buffer (Roche), with 10% phosphor STOP and 10% protease inhibitor cocktail. Cell lysates were then collected after centrifugation at 12,000 rpm for 10 m at 4°C. 30μg of lysate protein was loaded and total cellular protein was separated with 8% SDS-polyacrylamide gel electrophoresis, then transblotted onto NC membrane (Life Technology). The membrane was probed with anti-ZIP4 (Proteintech, 1:2000), ZO-1, claudin-1, ZEB1(Cell signaling Technology 1:500), pFAK, FAK (Abcam, 1:1000), pPaxillin, Paxillin (Life tech, 1:1000), and anti-β-actin (Abcam, 1:10000) antibody at 4 °C overnight, and then washed three times with 0.1% Tween 20-TBS and incubated with horseradish peroxidase-linked or NIR-coupled secondary antibody (1:5000) for 2 h at room temperature. The membrane was washed three times with 0.1% Tween 20-TBS. The immunoreacted bands were detected using an enhanced chemiluminescent (ECL) plus reagent kit. We are using the Li-COR Odyssey Fc machine to detect both ECL and the NIR-labeled 2^nd antibodies.

Human pancreatic adenocarcinoma and surrounding benign tissues were collected and processed into 5-μm slides. Fixed tissue slides were incubated in 3% hydrogen peroxide solution to quench endogenous peroxidase activity for 15 m and were subsequently washed with PBS. The slides were then incubated in blocking buffer for 30 m at room temperature and stained with anti-hZIP4 antibody (Proteintech, 1:500), ZO-1 (NOVUS,1:100), claudin-1 (1:100, Cell Signaling Technology), ZEB1 (1:250, Sigma) and incubated overnight at 4°C. After washing with PBS, the section was incubated with polymer secondary antibody for 30 m (Vector Laboratories). Immune complexes were detected with diaminobenzidine (DAB) under a phase-contrast microscope. The sections were then mounted and observed under a phase-contrast microscope. The slides were scanned using Aperio scanning software and the positivity was analyzed automatically. The KPC mouse sections were obtained from Dr. Courtney Houchen. ZIP4, ZO-1 (Proteintech), claudin-1 (Proteintech), ZEB1 (Sigma), pFAK (Abcam) and pPaxillin (Lifetech) were stained in pancreatic cancer tissue of KPC mouse sections as above.

AsPC-1 and Panc-1 cells were fixed with 4% PFA, blocked with PBS containing 4% BSA for 30 m at room temperature, and then incubated overnight at 4°C with the primary antibodies for ZIP4 (Proteintech), ZO-1, and claudin-1 (Cell Signaling Technology) diluted in the same buffer. After three washes, slides were incubated at room temperature for 1 h with secondary antibodies (1:200) and Hoechst (1 μg/ml) and were mounted with Prolong diamond antifade mounting (Life Technology). Sections were photographed using EVOS FLC (Life Technology).