human her2 wildtype  (Addgene inc)

Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet: Biosensors fused to HER2-Nb precisely report [K + ] ex changes upon immobilization on HER2 expressing HEK293 cells (A) Coomassie-stained SDS PAGE of 1 μg protein (left panel) and immunoblot analysis using anti-VHH antibody (right panel) of purified HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon proteins are shown. (B and C) Representative confocal images of living HEK293 cells transiently overexpressing HER2 (upper row) or untransfected HEK293 cells (lower row) following incubation with HER2-Nb-GEPII 1.0 (B) or HER2-Nb-pH-Lemon (C) are shown. Scale bar 10 μm, N= 4. (D) Response of HER2-Nb-GEPII 1.0 immobilized on HEK293 cell transiently overexpressing HER2 in response to buffers with different K + . Shown is a representative measurement of four cells (mean ± SD in red, traces from individual cells in black). (E) Respective single wavelength traces (FRET in red, mseCFP in pink) of the ratio curve as shown in (D) in response to K + alterations.

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Expressing, Staining, SDS Page, Western Blot, Purification, Incubation

Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet: HER2-Nb-biosensors specifically label endogenous HER2 on HER2 positive breast cancer cells (A) Schematic illustration of a HER2 positive SkBr3 cell endogenously expressing HER2 on the cell surface. Biosensors fused to HER2 can be bound to HER2 for immobilization on the plasma membrane. Figure created using BioRender. (B) Representative images of SkBr3 cells following incubation with HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon. Scale bar 10 μm, n= 4 experiments representing biological replicates. (C) Representative confocal images of HER2 negative MCF7 breast cancer cells following incubation with HER2-Nb-GEPII 1.0 and HER2-Nb-pH-Lemon are shown. Scale bar 10 μm, N= 4. (D) Determination of cell viability of SkBr3 cells using MTT in response to vehicle (ctrl), unfused HER2-Nb and HER2-Nb-GEPII 1.0 after 3, 6, 24, and 48 h after immobilization. Not significant as determined using one-way ANOVA, Dunn’s Multiple Comparison Test.

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Expressing, Clinical Proteomics, Membrane, Incubation, Comparison

Journal: iScience

Article Title: Monitoring extracellular ion and metabolite dynamics with recombinant nanobody-fused biosensors

doi: 10.1016/j.isci.2022.104907

Figure Lengend Snippet:

Article Snippet: For immobilization of SPOT-Nb-sensors on HEK293 cells, the cells were transfected with a plasmid encoding a GPI-anchored SPOTtag (GPI-SPOT) using Lipofectamine 2000 reagent (Thermo Fisher) according to the manufacturer's instructions 2 days before experiments, followed by the removal of the transfection reagent after 6 h. For transient HER2 overexpression in HEK293 cells, the cells were transfected with a plasmid encoding human HER2 wildtype ( )(Addgene plasmid #16257) the day before measurement with removal of the transfection reagent after 6 hours.

Techniques: Virus, Recombinant, Staining, Plasmid Preparation, Protease Inhibitor, Software