D110087

Bacterial samples were resuspended in 1× protein loading buffer and boiled at 98 °C for 8 min. 0.05 OD of proteins samples were loaded each lane. Proteins were transferred onto PVDF membranes (#10600023, Cytiva) for 45 min at 150 V in transfer buffer. Membranes were blocked for 1 h at room temperature in 1× TBST buffer with 5% (w/v) skim milk (#A600669-0250, Sangon), washed with TBST twice and incubated with monoclonal α-FLAG (Sigma-Aldrich #F1804-5MG; 1:10,000), α-SipC (1: 3,000) or α-GroEL (Sigma-Aldrich #G6532; 1:10,000) antibodies for 1 h at room temperature. After three TBST washes, membranes were incubated with secondary α-mouse or α-rabbit HRP-linked antibodies (Sangon #D110087 or #D110058; 1:10,000) for 1 h at room temperature. Chemiluminescence was developed using the high sensitive ECL luminescence reagent (#C500044-0100, Sangon), and then visualized on ChemiScope 6000SE and quantified using ImageJ Software.

In brief, the protein samples were separated by SDS-PAGE using a 4% stacking gel and an 8 ~ 10% main gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% milk at room temperature for 1–2 h and then incubated with anti-SIRPα (dilution 1:1000, AB8120, Abcam), anti-PD-1 (dilution 1:200, ab52587, Abcam) or anti-Phosphotyrosine (dilution 1:1000, 05–321, Millipore) dissolved in primary antibody dilution buffer overnight at 4 °C. Then the membrane was washed and incubated with appropriate HRP-coupled goat anti-rabbit IgG (D110058, Sangon Biotech) and HRP-coupled goat anti-mouse IgG (D110087, Sangon Biotech) for 1–2 h at room temperature. Finally, the membranes were exposed using an enhanced chemiluminescence (ECL) detection kit (P1030, Applygen). Results were analyzed by ImageJ software (NIH, Bethesda, MD, United States).

The paraffin sections of esophageal cancer and adjacent nontumor tissues (HEsoS160CS01, Shanghai Outdo Biotech Co., Ltd.) were deparaffinized with xylene and hydrated with absolute ethanol, 95% ethanol, 80% ethanol, 70% ethanol and distilled water, respectly. After 30 min microwave heat recovery by citric acid antigen retrieval method, the sections were cooled to room temperature and washed with PBS (0.01 M, pH 7.4), and then blocked with 2% BSA blocking solution at room temperature for 2 h. After blocking, the sections were incubated with the primary MUC13 (1: 50, Santa Cruz, USA) antibody for 2 h at 37 °C. Then rinsed three times in PBS, followed by incubation of the secondary antibody (D110087,Sangon Biotech Co., Ltd) at 37 °C for 2 h. DAB substrate kit was used to perform the chromogenic reaction. During which, slices were observed under the microscope to confirm the color end point. Then, the nucleus were counterstained with hematoxylin for 3 min. The slides were dehydrated in a series of graded ethanol solutions and transparented with xylene, and then mounted with a neutral gum. Finally, semiquantitative analysis of immunohistochemical results was conducted by Image ProPlus software, and positive cell percentages were calculated using IHC plug-in after color deconvulutionfor H&E DAB.