Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 3. Requirement of the ligand-binding domain of Notch1 for the activation and increased cell surface expression of Notch1 by transient co-transfection of TM2D3. (a, b) Activation of Notch1 with a full-length ectodomain but not those that lack EGF repeats (Notch1 LNR, LNR CC > SS, and ΔE) (Supplementary Fig. S5 online) by co-transfection of TM2D3. Cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoblotting was performed with the indicated antibodies. Notch1 LNR CC > SS and ΔE are constitutively active. (c) Inability of TM2D3 to activate Notch1 that lacks EGF repeats 11 and 12. Experiments were conducted as in a and b. (d, e) Increased Notch1 expression at cell surface by co-transfection of TM2D3 and its independence on EGF repeats 11 and 12. Cells transfected with vectors for the indicated proteins or empty vector (Vector) as a control were incubated with or without a non-membrane permeable biotinylation reagent as indicated. After fractionation with an avidin agarose, immunoblotting was performed with the indicated antibodies. (f) Cell surface expression of TM2D3. Experiments were conducted as in (d) and (e).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Cotransfection, Transfection, Plasmid Preparation, Control, Western Blot, Incubation, Membrane, Fractionation, Avidin-Biotin Assay

Journal: Scientific reports

Article Title: TM2D3, a mammalian homologue of Drosophila neurogenic gene product Almondex, regulates surface presentation of Notch receptors.

doi: 10.1038/s41598-023-46866-7

Figure Lengend Snippet: Figure 4. Physical association of Notch1 and TM2D3. (a) Physical association of TM2D3 and Notch1. 293 T cells were transfected with vectors for the indicated proteins or empty vector (Vector) as a control. Immunoprecipitation (IP) and immunoblotting were performed with the indicated antibodies. (b, c) Requirement of NRR of Notch1 for physical association with TM2D3. Experiments were conducted as in (a). (d) Requirement of LNR-A of NOTCH1 for physical association with TM2D3. A schematic diagram of NOTCH1 constructs used is shown in Supplementary Fig. S5 online. Experiments were conducted as in a. (e) Requirement of C-terminal portion including transmembrane 2 domain of TM2D3 for the activation of Notch1. Experiments were conducted as in (a). (f) No dependency on any one region of TM2D3 for the physical association with Notch1. Experiments were conducted as in (b).

Article Snippet: An antibody against the C-terminus of human NOTCH1 (sc-6014), an antibody against the ECD of murine Notch1 (8G10; sc-32756), and antibodies against the ECD of Notch2 (sc-518169 and sc-5545) were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Western Blot, Construct, Activation Assay