Ab7755

To examine protein expression of C/EBPβ and YY1 in GE1 cells, immunofluorescence was carried out. After plating 30 μL of GE1 cells from confluence in 75 cm² flask on culture slides (Falcon^® CultureSlides, Corning^® BioCoat™, Hampton, NH, USA), the plates with cells were incubated at 33 °C for 12 h. After treatment with PgLPS for 12 or 24 h, the cells were rinsed with PBS twice and fixed by 4% paraformaldehyde for 15 min at RT. The fixed cells were rinsed by PBS twice and permeabilized with 0.5% Triton X‐100 in PBS for 10 min at RT. After removing permeabilization solution, there followed blocking with 1% bovine serum albumin in PBS for 20 min at RT. The cells were treated with 100 μL of primary antibodies diluted with buffer (1% BSA in PBS) for 1 h at RT followed by incubation with secondary antibodies for 1 h at RT. For the primary antibodies, we used C/EBPβ (1 : 50, sc‐746; Santa Cruz Biotechnology), YY1 (ChIP Grade; ab38422; Abcam) rabbit polyclonal antibodies and CK19 (1 : 100, ab7755, Abcam) mouse monoclonal antibody. For detection signaling for fluorescence, we used goat anti‐mouse IgG H&L (Alexa Fluor^® 488, 1 : 200, ab150113, Abcam) and goat anti‐rabbit IgG H&L (Alexa Fluor^® 647, 1 : 200, ab150079, Abcam). Mounting and nuclear staining with 4′,6‐diamidino‐2‐phenylindole (DAPI) were performed (Fluoroshield mounting medium with DAPI, ab104139, Abcam).

Total proteins extracted from Ca9‐22 cells were separated by 12% SDS/PAGE and transferred onto a Hybond 0.2‐μm PVDF membrane. The membrane was incubated with anti‐AMTN (ab122312; Abcam, Cambridge, UK), anti‐CK19 (ab7755; Abcam), and anti‐α‐tubulin (sc‐5286; Santa Cruz Biotechnology, CA, USA) antibodies for 2 h. Anti‐rabbit and anti‐mouse IgG conjugated with horseradish peroxidase were used as the secondary antibodies. Immunoreactivities were detected by ECL Prime Western Blotting Detection Reagents 24.

IHC assays for CD8 (ZA-0508; ZSGB-BIO, City of Beijing, China), CD133 (ab226355; Abcam, City of Cambridge, USA), CK19 (ab7755; Abcam) PD-1 (ab52587; Abcam), and Tim-3 (ab241332; Abcam) were performed on pancreatic cancer tissues using and IHC kit (ZLI-9018; ZSGB-BIO, City of Beijing, China) in accordance with standard protocols following a previously described procedure^{25 (link)}.