Rabbit anti gata6

Paraffin-embedded gastric cancer tissue sections of selected cases from the above-mentioned patients were dewaxed in xylene and rehydrated in alcohol. Antigen retrieval was performed by heating each section at 100 °C for 25 min in 1× Novocastra^TM Epitope Retrieval Solution (pH = 9.0, Leica Biosystems, Newcastle, UK). After 5 min rinses in phosphate-buffered saline (PBS), we followed the protocol of Novolink^TM MaxPolymer Detection System (Leica, Wetzlar, Germany) to stain patient samples, with rabbit anti-GATA6 (1:50, Abcam), rabbit anti-TFF1 (1:50, Abcam), or mouse anti-TFF2 (1:50, Abcam) at 4 °C, overnight, followed by secondary antibody containing post primary reagent and HRP-linked polymer. Slides were finally counterstained with hematoxylin.

The lysates of fresh tissues and the H9-derived cells were subjected to IB assay according to the previous protocol (Liu et al., 2012 (link); Tripathi and Mishra, 2012 (link)). Then the proteins were detected using indicated antibodies. Antibodies used for IB are as follows: mouse anti-GFAP (1:500, Proteintech, Chicago, IL, USA), mouse anti-NEUN (1:500, Millipore, Belecula, CA, USA), rabbit anti-BAF45D (1:500, Proteintech, Chicago, IL, USA), rabbit anti-GATA6 (1:100, Abcam, New Territories, HK), mouse anti-OCT4 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PAX6 (1:500, Millipore, Belecula, CA, USA) and rabbit anti-GAPDH (1:2000, Proteintech, Chicago, IL, USA) antibodies. More descriptions of the antibodies used for IB were shown in Table S1.

Cells were lysed with 100 μL of PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) and protein concentrations determined by Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. Samples and pre-stained protein markers were electrophoresed through 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, and then transferred to polyvinylidene fluoride (PVDF) membranes, using the Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad). The membrane was then incubated overnight at 4 °C with primary antibodies, rabbit anti-pSTAT3 (1:1000, Cell Signaling, Beverly, MA, USA), mouse anti-STAT3 (1:1000, Cell Signaling), rabbit anti-GATA6 (1:1000, Abcam, Cambridge, UK), rabbit anti-TFF1 (1:1000, Abcam), mouse anti-TFF2 (1:1000, Abcam) or mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), as diluted in 1× phosphate-buffered saline with Tween (PBST). The membranes were incubated at room temperature for 1 h, with secondary antibodies (Thermo Fisher, anti-mouse, 1:1000 or anti-rabbit 1:1000, diluted in 1× PBST). Proteins were detected by an enhanced chemiluminescence horseradish peroxidase (HRP) substrate detection kit (Merck Millipore) and MiniChemi^TM Chemiluminescence imaging system (Sage Creation, Beijing, China).