Anti gbe1

The protocols used for IHC and immunofluorescence were performed according to previous studies [22 (link)]. Anti-GBE1 (1:300; Abcam, USA), anti-PD-L1 (1:300; Cell Signaling Technology, USA), CD8 (1:300; Abcam, USA), CCL5 (1:300; BioLegend, USA), CXCL10 (1:300; Ruiyingbio, China) were used as primary antibodies. For IHC, three fields of view per sample were imaged. The intensity of immunostaining was taken into consideration when analyzing the data. The percentage scoring of immunoreactive tumor cells was as follows: 0 (< 10%), 1 (10–40%), 2 (40–70%), and 3 (> 70%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (yellowish), 2 (light brown), 3 (dark brown). The intensity of staining was obtained by multiplying the two items into a total score, and the scores ranged from 0 to 9. In immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (BioLegend, California, USA, 1:500) were used to detect the primary antibodies. Nuclear staining was performed with DAPI (1:1000; Roche, USA). The samples were visualized with a fluorescence microscope (Olympus, IX71, Japan).

Immunohistochemistry and immunofluorescence were performed as previously described^{48 (link)}. Anti-GBE1 (1:300; Abcam), anti-Ki-67 (1:300; Abcam), anti-caspase 3 (1:300; Abcam), and anti-HIF1α (1:300; Proteintech, Rosemont, IL, USA) were used as primary antibodies. For immunofluorescence, Cy3- and FITC-conjugated secondary antibodies (1:500; BioLegend, San Diego, CA, USA) were used to detect primary antibodies. The samples were then imaged using a fluorescence microscope (IX71; Olympus, Japan).

Cells were extracted into cold lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM MgCl₂, 0.5% Triton X-100, phosphatase inhibitor mix (1 mM NaF, 1 mM Na₃VO₄, and 1 mM β-glycerol phosphate), and protease inhibitor mix (1 mM PMSF, 2 μg/ml Roche protease inhibitor cocktail (aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin A). The protein concentration was determined by using the BCA method (Biyuntian, China). The following primary antibodies were used: anti-GBE1 (Abcam, USA), anti-TMEM173 (ProteinTech Group, USA), anti-PD-L1 (Cell Signaling Technology, USA), and β-actin (Santa Cruz Biotech, USA) as control. These primary antibodies were detected with horseradish peroxidase-conjugated anti-IgG, and the detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit (Pierce, USA). The band images were digitally visualized and quantified with a Fluor Chem FC2 imaging system (Alpha Innotech, USA).