y1284  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: Selectivity of receptor activation and downstream signaling by single ligands correlate in barcoded assays (A) Heatmap showing stimulation profiles on ERBB receptors, HTR2A, and downstream signaling pathways. Assays for receptors were performed using barcoded split TEV, assays for signaling pathways with pathway sensors coupled to barcodes. Compound effects are shown as log2-transformed fold change. (B–H) Barcoded assays align with luciferase readouts. Visualization of selected data from (A), comparing barcoded assays (black) with luciferase assay readouts (red). Assays for receptors were performed using split TEV, assays for signaling pathways with pathway sensors. Dose response graphs for EGFR (B), ERBB4 (B), EGR1p only (D), and EGR1p and ERBB4 transfected (E), HTR2A (F), CRE and HTR2A transfected (G), and NFAT and HTR2A transfected (H) with single stimuli applied at increasing concentrations. EGFld, EGF-like domain. Error bars represent SEM, n = 3 for barcoded assays, and n = 6 for luciferase assays. See also Figure S2 and Table S2 .

Article Snippet: Phosphorylation levels of EGFR and ERBB4 were assayed using p -EGFR-Y1068 (RRID: AB_2096270 ) (clone D7A5, dilution 1:500, No. 3777, Cell Signaling Technology) and p -ERBB4-Y1284 antibodies (RRID: AB_2099987 ) (clone 21A9, dilution 1:500, No. 4757, Cell Signaling Technology).

Techniques: Activation Assay, Protein-Protein interactions, Transformation Assay, Luciferase, Transfection

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: The barcoded ERBBprofiler reveals known and previously uncharacterized selectivity properties of ERBB receptor antagonists (A) Heatmap showing antagonistic effects of compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells. Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways with barcoded pathway sensors. In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 μM). Compound effects are shown as log2-transformed fold change. (B–E) Dose response graphs comparing drug selectivity for receptors EGFR and ERBB4 (B, D) and downstream MAPK signaling (C, E) of compounds AG1478 (B, C), and pyrotinib (D, E). Data was extracted from the heatmap shown in (A). n = 3. (F–I) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for AG1478 (F), osimertinib (G), poziotinib (H), and pyrotinib (I). In addition to the increasing concentrations of the compounds shown, all assays contained the constant stimulation mix as in (A). (J–M) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. and GloSensor, respectively, as readouts in PC12 cells treated with increasing concentrations of AG1478 (J), osimertinib (K), poziotinib (L), and pyrotinib (M). As in luciferase assays, the constant stimulation mix was constantly present, next to the mentioned compounds. Error bars represent SEM, n = 3 for barcode assays (B-E), n = 6 for luciferase, Fluo-4 a.m., and GloSensor assays (F-M). See also Figure S3 , Tables S3 and .

Article Snippet: Phosphorylation levels of EGFR and ERBB4 were assayed using p -EGFR-Y1068 (RRID: AB_2096270 ) (clone D7A5, dilution 1:500, No. 3777, Cell Signaling Technology) and p -ERBB4-Y1284 antibodies (RRID: AB_2099987 ) (clone 21A9, dilution 1:500, No. 4757, Cell Signaling Technology).

Techniques: Protein-Protein interactions, Transformation Assay, Luciferase

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: Pyrotinib reveals selectivity for ERBB4 over EGFR (A and B) Dose response assays comparing AG1478 selectivity for receptors EGFR and ERBB4 (A) and downstream MAPK signaling (B) using firefly luciferase assays in PC12 cells. Assays for receptors were performed using split TEV, assays for MAPK signaling were conducted with an EGR1p pathway sensor. In addition to the increasing concentrations of AG1478, EGFR and ERBB4 assays contained a constant concentration of EGF (30 ng/mL) or EGF-like domain (10 ng/mL), respectively. (C and D) Western blot analyses of p -EGFR (in A549 cells) (C) and p -ERBB4 (in T-47 cells) (D) using increasing concentrations of AG1478. (E and F) Quantification of (C) and (D). (G and H) Dose response assays comparing pyrotinib selectivity for receptors EGFR and ERBB4 (G) and downstream MAPK signaling (H) using firefly luciferase assays in PC12 cells. Assays were conducted as in (A, B). (I and J) Western blot analyses of p -EGFR (in A549 cells) (I) and p -ERBB4 (in T-47 cells) (J) using increasing concentrations of pyrotinib. (K and L) Quantification of (I) and (J). Error bars represent SEM, n = 6 for luciferase assays (A, B, G, H), n = 3 for Western blot assays (E, F, K, L).

Article Snippet: Phosphorylation levels of EGFR and ERBB4 were assayed using p -EGFR-Y1068 (RRID: AB_2096270 ) (clone D7A5, dilution 1:500, No. 3777, Cell Signaling Technology) and p -ERBB4-Y1284 antibodies (RRID: AB_2099987 ) (clone 21A9, dilution 1:500, No. 4757, Cell Signaling Technology).

Techniques: Luciferase, Concentration Assay, Western Blot

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet: The barcoded ERBBprofiler reveals novel ERBB4 selective antagonists (A) Heatmap showing antagonistic effects of LDC compounds on ERBB receptors, HTR2A, and downstream signaling pathways in PC12 cells. Assays for receptors were performed using barcoded split TEV assays, assays for signaling pathways using barcoded pathway sensors. In addition to the increasing concentrations of the compounds shown, all assays contained constant concentrations of EGF (30 ng/mL), EGF-like domain (10 ng/mL), and serotonin (1 μM). (B–E) Dose response graphs comparing drug selectivity for ERBB4 over EGFR (measured with split TEV) (B, C) and downstream MAPK signaling (measured with the EGR1p sensor) (D, E) of compound A (B, D), and compound B (C, E). Data was extracted from the heatmap shown in (A). (F–I) Orthogonal validation for compound B using Western blot analyses of p -EGFR and p -ERK1/2 (measured in A549 cells) (F) and p -ERBB4 and p -ERK1/2 (measured in T-47 cells) (G) using increasing concentrations of compound B (Cpd B). (H) Quantification of relative p -EGFR and p -ERBB4 from (F, G). (I) Quantification of relative p -ERK1/2 from (F, G). (J and K) In vitro kinase activity assays using LANCE assays for compound A (J) and compound B (K) showing dose response graphs comparing drug selectivity for ERBB4 (red) and EGFR (black). (L) Dose response graphs for CRE sensor responses in PC12 cells using luciferase as readout for compounds A (black) and B (red). In addition to the increasing concentrations of the compounds, the constant stimulation mix as in (A) was present. (M and N) Dose response graphs for calcium and cAMP assays using Fluo-4 a.m. (black) and GloSensor (red), respectively, as readouts in PC12 cells treated with increasing concentrations of compound A (M) and compound B (N). In addition to the compounds, the constant stimulation mix was present as in (A). Error bars represent SEM; n = 3 for barcode assays (B–E), Western blots (F–I) and in vitro kinase assays (J, K); n = 6 for luciferase assays, Fluo-4 a.m., and GloSensor assays (L–N). See also Figures S4 and .

Article Snippet: Phosphorylation levels of EGFR and ERBB4 were assayed using p -EGFR-Y1068 (RRID: AB_2096270 ) (clone D7A5, dilution 1:500, No. 3777, Cell Signaling Technology) and p -ERBB4-Y1284 antibodies (RRID: AB_2099987 ) (clone 21A9, dilution 1:500, No. 4757, Cell Signaling Technology).

Techniques: Protein-Protein interactions, Biomarker Discovery, Western Blot, In Vitro, Activity Assay, Luciferase

Journal: iScience

Article Title: Profiling of ERBB receptors and downstream pathways reveals selectivity and hidden properties of ERBB4 antagonists

doi: 10.1016/j.isci.2024.108839

Figure Lengend Snippet:

Article Snippet: Phosphorylation levels of EGFR and ERBB4 were assayed using p -EGFR-Y1068 (RRID: AB_2096270 ) (clone D7A5, dilution 1:500, No. 3777, Cell Signaling Technology) and p -ERBB4-Y1284 antibodies (RRID: AB_2099987 ) (clone 21A9, dilution 1:500, No. 4757, Cell Signaling Technology).

Techniques: Virus, Recombinant, Biomarker Discovery, Software