D110058

One hundred thirty-one GC tissues and twenty-four matched adjacent normal tissues were embedded in paraffin according to standard procedures. All tissue blocks were cut into 4 µm sections and then deparaffinized and hydrated on the basis of standard procedures. The endogenous peroxidase activity of tissue sections was blacked by using 3% H₂O₂ for 15 min at room temperature. Sections were incubated in a wet chamber with the primary antibody TAF15 (1:100, ab134916, Abcam, USA) at 4 °C for overnight, and then incubated with the secondary antibody HRP goat anti-rabbit IgG (1:100, D110058, Sangon Biotech, Shanghai, China) for 1 h at room temperature. Finally, sections were developed using diaminobenzidine and haematoxylin. The images were reviewed by two pathologists in a blinded fashion. The staining percentage was scored as follows: 0 (< 5% positive cells); 1 (5–24% positive cells); 2 (25–49% positive cells); 3 (50–74% positive cells) and 4 (≥ 75% positive cells). The staining intensity was scored as follows: 0 (no staining), 1 (weak), 2 (moderate), and 3 (strong). The combined staining score was equivalent to the staining percentage score × staining intensity score and a combined score ≥ 4 was defined as positive expression, while a combined score < 4 was considered negative expression.

About 100 oocytes were lysed in the RIPA buffer supplemented with the protease inhibitor cocktail from Beyotime Institute of Biotechnology (P0013B and P1006; Shanghai, China). The target protein was first separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. Next, the membrane was blocked in TBST containing 5% BSA for 1 hour at room temperature, followed by incubation overnight at 4°C with anti‐CAT rabbit polyclonal antibody (1:200), anti‐BAX rabbit polyclonal antibody (1:500) and anti‐ACTB rabbit polyclonal antibody (1:1000). After washing 3‐5 times in TBST for 5‐10 minutes, the membrane was incubated with HRP‐conjugated goat anti‐rabbit IgG (1:2000) for 1 hour at 37°C in the dark. All four antibodies were purchased from Sangon Biotech Co., Ltd. (D122036, D120073, D110001 and D110058; Shanghai, China).

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).

Proteins isolated from SCs were used for Western blot analysis in accordance with previous standard methods [36 (link),37 (link)]. Proteins from SCs in each treatment group were separated through 10% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4 °C with primary antibodies (Table 1), rinsed three times with TBST, and incubated for 2 h at 37 °C with secondary antibodies (Sangon Biotech, D110058) in TBST. Related proteins were detected using AlphaImager^® (ProteinSimple, 92-13824-00, San Jose, CA, USA) HP. The intensity of all bands was quantified with GAPDH as the internal control using ImageJ software.