Additional file 1 of Quantitative assays for the measurement of HER1-HER2 heterodimerization and phosphorylation in cell lines and breast tumors: applications for diagnostics and targeted drug mechanism of action
Creators: name: DeFazio-Eli, Lisa
name: Strommen, Kristi
name: Dao-Pick, Trang
name: Parry, Gordon
name: Goodman, Laurie
name: Winslow, John
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences

description: Additional file 1: Supplementary Figures S1 to S6 Figure S1. Ligand-dependent and ligand-independent HER1-HER2 heterodimerization depends on receptor levels. A total of 293 cells were transfected with HER1 to generate 293H1_clone11. Clone11 was then co-transfected with HER2 to generate clones 12, 15, 16, and 19. Gray bars represent signal from mock-stimulated cells, and black bars represent signal from cells stimulated with 100 nM EGF. A) HER1 total lysate assay. All clones had the same amount of HER1. B) HER2 total lysate assay. Clones displayed different amounts of total HER2. C) HER1-HER2 heterodimer lysate assay. Ligand-dependent and ligand-independent HER1-HER2 heterodimer formation increased with increasing HER2 receptor number in clones with the same amount of HER1. Figure S2. Specificity of clone 53A5 for phospho-HER1 in FFPE assay. AU565 FFPE slides from cells were mock-stimulated or stimulated with 100 nM EGF for 10 minutes. The pHER1-HER2 FFPE assay was performed, but peptide was incubated with antibody in molar ratios 1:0, 1:1, 1:10, or 1:100 of antibody to peptide. Gray bars represent signal from mock-stimulated cells, and black bars represent signal from EGF-stimulated cells. Phospho-HER1-HER2 signal is competed to basal levels using the antigenic peptide H1pY1173 but not with the nonphosphorylated H1Y1173 nor the homologous HER2 peptides H2pY1248 nor H2Y1248. Figure S3. Effects of lapatinib and erlotinib measured by VeraTag™ lysate assays in H1650. H1650 cells were serum-starved overnight then treated with lapatinib or erlotinib for two hours at the indicated concentrations, in units of μM. A final concentration of 16 nM EGF was added or mock-added to the drug-containing media for 10 minutes prior to harvesting cells for lysate or FFPE. VeraTag™ lysate assays were used to examine A) effects of erlotinib and B) effects of lapatinib on HER1-HER2 heterodimerization (top row), phosphorylation of HER2 (second row), phosphorylation of HER1 (third row), and HER1-HER1 homodimerization (bottom row). Figure S4. Comparison of two different formats of a HER1 total protein VeraTag™ assay performed in HER2+ breast FFPE tumors. A) Comparison of a HER1 ICD-directed total protein assay with a HER1 ECD-directed total protein assay, both applied to intact FFPE tumor sections. B) Comparison of a HER1 ICD-directed total protein assay performed in intact FFPE tumor sections with a HER1 ECD-directed total protein assay having non-tumor elements macrodissected from the FFPE tumor sections. C) Comparison of HER1 values measured in intact versus macrodissected FFPE tumor sections assayed by the HER1 ECD-directed total protein assay. Figure S5. Comparison of HER1 ECD-directed total protein assay with HER1 IHC H-score. A) A subset of 30 HER2+ breast tumors was assayed by a HER1 ECD-directed total protein assay with non-tumor elements macrodissected away, and the HER1 levels were compared with the tumor cell HER1 IHC H-scores measured on adjacent sections. The IHC H-score spans 0 to 300. Tumors with an H-score of 0 were assigned a score of 0.1 to make plotting on a log scale possible. B) The percentage of the 30 tumors at each HER1 IHC test category. C-F) Micrographs of the HER1 IHC staining representing four separate invasive ductal carcinoma breast tumors that are representative of other tumors having similar staining intensity from IHC = 3+ to 0, respectively (4× magnification). G-J) Same tumor micrographs as in C-F but at 20× magnification. Figure S6. Western blot and co-IP/Western blots of lysed tumor tissue homogenates. Fresh frozen tumors matched with FFPE blocks of HER2+ breast tumors were lysed and homogenized with protease and phosphatase inhibitors, and analyzed by SDS-PAGE and Western blots. Sections of the matched FFPE blocks were assayed by the comparable VeraTag™ assay. A) HER2 Western blot comparison of tumor lysate with HER2 total protein VeraTag™ assay. B) HER1 Western blot of tumor lysate comparison with HER1 ECD-directed total protein VeraTag™ assay. C) Tumor lysate immunoprecipitated with a HER2 antibody and Western blot with a pan-phosphotyrosine antibody comparison with pHER2 VeraTag™ assay. D) Tumor lysate immunoprecipitated with pHER1(1173) antibody and Western blot with a HER2 antibody comparison with pHER1-HER2 VeraTag™ assay. (PDF 621 KB)