Antigbp2

IHC staining was conducted on the above sections according to the standardized procedures. Sections were retrieved by EDTA. The primary antibodies used were as follows: antiGBP2 (1:3000 dilution, Cat. 11854-1-AP, ProteinTech), antiPD-L1 (Ready-to-use, Cat. GT2280, GeneTech), antiPD-1 (Ready-to-use, Cat. GT2281, GeneTech), antiCD8 (Ready-to-use, Cat. GT2112, GeneTech), antiMLH1 (Ready-to-use, Cat. GT2304, GeneTech), antiMSH2 (Ready-to-use, Cat. GT2310, GeneTech), antiMSH6 (Ready-to-use, Cat. GT2195, GeneTech), and antiPMS2 (Ready-to-use, Cat. GT2149, GeneTech). Staining was visualized with DAB and hematoxylin counterstain, and stained sections were captured using Aperio Digital Pathology Slide Scanners. For semi-quantitative analysis, the stained sections were independently evaluated by two pathologists. GBP2 and PD-L1 were assessed by according to the evaluation standard on a 12-point scale by calculating the immunoreactivity score (IRS) [31 (link)]. CD8 and PD-1 were assessed by estimating the percentage of cells with strong intensity of membrane staining in the stromal cells.

Antibodies against Irgb6 (T-cell specific GTPase [TGTP]; sc-11079), Irgm1 (LRG-47; sc-11075), Irgm2 (GTPI; sc-11088), and Irgm3 (inducibly expressed GTPase [IGTP]; sc-136317) were purchased from Santa Cruz Biotechnology, Inc. Antibodies against FLAG M2 (F3165), β-actin (A1978), and HA were obtained from Sigma-Aldrich. An Anti-HA 1.1 mouse monoclonal antibody was purchased from BioLegend. Rabbit polyclonal anti-GBP2 and mouse monoclonal anti-p62 (PM045) antibodies were obtained from Proteintech and MBL International, respectively. An anti-ubiquitin mouse monoclonal antibody (FK2; MFK-004) was obtained from Nippon Biotest Laboratories. A mouse monoclonal anti-Irga6 (10D7) antibody was provided by Dr. JC Howard (Instituto Gulbenkian de Ciencia). A rabbit polyclonal anti-GBP1 antibody was provided by Dr JC Boothroyd (Stanford University School of Medicine). A mouse monoclonal anti-GRA2 antibody were provided by Dr. D Soldati-Favre (University of Geneva). A custom anti-Irgm2 antibody was purchased from Cosmobio for microscopy analysis. Recombinant mouse IFN-γ was purchased from PeproTech.