Immunohistochemistry and Western blotting protocols

Basic IHC and IF procedures in FFPE sections (4 μm) were detailed previously^{11 (link),14 (link)–17 (link)}. For IHC, slides were deparaffinized in xylene and hydrated in gradient alcohols to water. Slides were treated with antigen retrieval in 10 mM citrate buffer (pH 6.0) and incubated with primary antibodies (Supplementary Table 1; optimal dilutions were determined by antibody titration experiments in pilot studies) followed by secondary antibodies and DAPI counterstaining (when applicable). IHC images were captured using an Olympus inverted (epifluorescence) microscope. The whole-mount and TMA IHC slides were scanned using Aperio ScanScope imaging system (Aperio Technologies, Vista, CA, USA) and a ×40 objective. Images were analyzed using the ScanScope software. For Western blotting analysis, whole cell lysate was prepared in RIPA buffer and run on 4–15% gradient SDS-PAGE gels. The proteins were transferred to nitrocellulose membrane followed by incubation with primary antibodies (Supplementary Table 1) and corresponding secondary antibodies. Films were developed using Western Lighting ECL Plus reagent (PerkinElmer). Representative original films for multiple Western blotting analyses were presented in Supplementary Figs. 20–25.