Cell surface expression of phosphate transporters was monitored on HEK293T cells with soluble ligands derived from the receptor-binding domain (RBD) of different retroviral Envs. RBD derived from mouse X-MLV, koala endogenous retrovirus, and mouse amphotropic-MLV Envs, were used to detect XPR1/SLC53A1, PiT1/SLC20A1 and PiT2/SLC20A2, respectively, and binding assays performed as previously described10 (link),35 (link),36 (link). Control expression of the GLUT1 glucose transporter was monitored with the GLUT1-specific H2RBD ligand, derived from the human T-cell leukemia virus type 2 Env30 (link),33 (link). Briefly, 5 × 105 cells were incubated in PBA (PBS complemented with 2% FBS) containing the adequate RBD, for 30 min at 37 °C under agitation, followed by two washes with PBA and incubation with an Alexa Fluor 488-conjugated anti-mouse IgG1 antibody (Life Technologies; 1:5000) for 20 min at 4 °C. Cells were promptly analyzed on NovoCyte flow cytometer (Becton Dickinson), and data were analyzed with the FlowJo package. All RBD ligands were produced as previously described10 (link),35 (link), or obtained from METAFORA-biosystems.
Alexa fluor 488 conjugated anti mouse igg1 antibody
Manufactured by Thermo Fisher Scientific
Alexa Fluor 488-conjugated anti-mouse IgG1 antibody is a fluorescently-labeled secondary antibody. It is designed to bind to mouse IgG1 primary antibodies, allowing for the detection and visualization of target proteins or other biomolecules in various immunoassay applications.
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2 protocols using alexa fluor 488 conjugated anti mouse igg1 antibody
1
Quantifying Phosphate Transporter Expression
2
Retroviral Receptor Expression Profiling
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Cell surface expression of phosphate transporters was monitored with soluble ligands derived from the receptorbinding domain (RBD) of different retroviral envelope glycoproteins. Production of and binding with RBD from X-MLV (XRBD), and koala retrovirus (KoRBD), or with a soluble ligand derived from the surface unit (SU) of amphotropic-MLV (ASU), used to detect XPR1, PiT1, and PiT2, respectively were performed as previously described [10, 15] . Briefly, 5 9 10 5 cells were resuspended in PBA (PBS with 2 % FBS) containing the adequate RBD and incubated for 30 min at 37 °C, followed by two washes with PBA and incubation with an Alexa Fluor 488-conjugated anti-mouse IgG1 antibody (Life Technologies; 1:5000) for 20 min at 4 °C. Cells were promptly analyzed on FACSCalibur instrument (Becton-Dickinson) and data were analyzed with the FlowJo package.
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