Journal: Developmental cell

Article Title: Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand

doi: 10.1016/j.devcel.2020.09.017

Figure Lengend Snippet: (A) Purified SCUBE2-SHH or SHH-N was added to HEK293T cells expressing EGFP-tagged PTCH1, and bound ligand was quantified by anti-SHH (5E1) immunofluorescence. SCUBE2 reduces SHH affinity for PTCH1. Data are normalized between background signal (untreated cells) and maximum SHH-N binding (defined as 100%), and are fit with a three-parameter curve. Points represent average binding for four replicates, and error bars represent SEM. At least 200 cells were measured per replicate.

Article Snippet: Purified mouse anti-SHH (5E1), anti-FLAG-M1 and anti-HPC monoclonal antibodies were fluorescently labeled using Alexa Fluor 568 NHS ester or Alexa Fluor 647 NHS ester (Thermo), according to the manufacturer’s instructions.

Techniques: Purification, Expressing, Immunofluorescence, Binding Assay

Journal: Developmental cell

Article Title: Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand

doi: 10.1016/j.devcel.2020.09.017

Figure Lengend Snippet: (A) Fluorescently labeled SCUBE2 (300 nM) was incubated with cells expressing EGFP-tagged SHH-binding proteins, and bound ligand was quantified by fluorescence microscopy. SCUBE2 binds to CDON/BOC, but not to other SHH interactors. Data are normalized between binding to EGFP-tagged SMO (negative control) and the highest bound signal (100%). Box plots represent median and the first and third quartiles of binding. At least 400 cells were measured per condition.

Article Snippet: Purified mouse anti-SHH (5E1), anti-FLAG-M1 and anti-HPC monoclonal antibodies were fluorescently labeled using Alexa Fluor 568 NHS ester or Alexa Fluor 647 NHS ester (Thermo), according to the manufacturer’s instructions.

Techniques: Labeling, Incubation, Expressing, Binding Assay, Fluorescence, Microscopy, Negative Control

Journal: Developmental cell

Article Title: Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand

doi: 10.1016/j.devcel.2020.09.017

Figure Lengend Snippet: (A) Fluorescently labeled SCUBE2 (600 nM), alone or in complex with SHH(HPC7) (300 nM), was incubated with cells expressing EGFP-tagged GAS1, scFv5E1::TM (positive control), or SMO (negative control), and bound SCUBE2 was measured by fluorescence microscopy. Even when complexed with SHH(HPC), SCUBE2 is not recruited to GAS1, in contrast to scFv5E1::TM. Data are normalized between binding of SCUBE2-SHH(HPC) to the negative and positive controls. At least 600 cells were measured per condition.

Article Snippet: Purified mouse anti-SHH (5E1), anti-FLAG-M1 and anti-HPC monoclonal antibodies were fluorescently labeled using Alexa Fluor 568 NHS ester or Alexa Fluor 647 NHS ester (Thermo), according to the manufacturer’s instructions.

Techniques: Labeling, Incubation, Expressing, Positive Control, Negative Control, Fluorescence, Microscopy, Binding Assay

Journal: Developmental cell

Article Title: Hedgehog Pathway Activation Requires Coreceptor-Catalyzed, Lipid-Dependent Relay of the Sonic Hedgehog Ligand

doi: 10.1016/j.devcel.2020.09.017

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Purified mouse anti-SHH (5E1), anti-FLAG-M1 and anti-HPC monoclonal antibodies were fluorescently labeled using Alexa Fluor 568 NHS ester or Alexa Fluor 647 NHS ester (Thermo), according to the manufacturer’s instructions.

Techniques: Recombinant, SYBR Green Assay, Purification, Luciferase, CRISPR, Sequencing, Blocking Assay, Software, Microscopy