Journal: STAR Protocols

Article Title: Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

doi: 10.1016/j.xpro.2026.104361

Figure Lengend Snippet: Preparation for PLA by optimization of antibody concentration and transfection efficiency (A) Varying antibody concentrations stain the overexpressed protein/-tag differentially well. This test is done to find the highest dilution to observe strong staining while limiting the amount of antibody used. Overexpression of SARS-CoV-2 E protein with a StrepII-tag. green, StrepII-AF488; blue, DAPI. Scale bar: 20 μm. (B) Optimization of the transfection using different transfection reagents, here Lipofectamine2000 or Lipofectamine3000, which affects the expression of the (viral) protein. Overexpression of SARS-CoV-2 E protein with a StrepII-tag. green, StrepII-AF488; blue, DAPI. Scale bar: 50 μm.

Article Snippet: Monoclonal mouse anti-StrepII-tag Antibody (517) (PLA 1:400) , Novus Biologicals , Cat#NBP2-43735.

Techniques: Concentration Assay, Transfection, Staining, Over Expression, Expressing

Journal: STAR Protocols

Article Title: Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

doi: 10.1016/j.xpro.2026.104361

Figure Lengend Snippet: Expected outcome of the PLA analysis of an overexpressed viral protein and an endogenous protein (A) Representative images of PLA protocol performed in cells either transfected with a vector control or a vector encoding for StrepII-tagged SARS-CoV-2 E T9 (wildtype) or the T9I variant. Cells are incubated with the individual antibodies targeted against StrepII-tag or the endogenous target ABCG2. Red, PLA; blue, DAPI. Scale bar: 20 μm (B) Quantification of the immunofluorescence images of (A). Lines represent the mean of N = 19-70 (individual cells) ± SEM.

Article Snippet: Monoclonal mouse anti-StrepII-tag Antibody (517) (PLA 1:400) , Novus Biologicals , Cat#NBP2-43735.

Techniques: Transfection, Plasmid Preparation, Control, Variant Assay, Incubation, Immunofluorescence

Journal: STAR Protocols

Article Title: Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

doi: 10.1016/j.xpro.2026.104361

Figure Lengend Snippet: Quantification of PLA dots per cell ROIs are defined using images of the co-staining of the overexpressed protein/-tag and saved in the ROI manager in ImageJ. These ROIs are applied to the binary PLA files and within these ROIs the particles are automatically counted using the analyze particle function of ImageJ. The count in the summary table (red box) indicates the number of PLA dots per ROI. Green, StrepII-tag-AF488; white, PLA dot images as binary files. Scale bar: 20 μm.

Article Snippet: Monoclonal mouse anti-StrepII-tag Antibody (517) (PLA 1:400) , Novus Biologicals , Cat#NBP2-43735.

Techniques: Staining

Journal: STAR Protocols

Article Title: Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

doi: 10.1016/j.xpro.2026.104361

Figure Lengend Snippet: Comparison of PLA quantification by ImageJ and Imaris (A) Quantification of the images shown in A using semi-automated counting in ImageJ. Only cells positively staining for StrepII-tag overexpression were included in the PLA dot quantification of cells transfected with constructs encoding for SARS-CoV-2 E T9 or E T9I. Lines represent the mean of N = 19-70 (individual cells) ± SEM. Cells in the vector control were not gated for StrepII expression. (B) Quantification of the images shown in A using bulk analysis with Imaris. PLA dots and DAPI-stained nuclei were automatically counted. Lines represent the mean of N = 19–24 (individual tiles) ± SEM after Outlier identification using ROUT method in GraphPad Prism. Student’s t test with Welch’s correction. ∗∗∗, p < 0.001.

Article Snippet: Monoclonal mouse anti-StrepII-tag Antibody (517) (PLA 1:400) , Novus Biologicals , Cat#NBP2-43735.

Techniques: Comparison, Staining, Over Expression, Transfection, Construct, Plasmid Preparation, Control, Expressing

Journal: STAR Protocols

Article Title: Protocol to differentially quantify spatially resolved viral protein-cellular protein interactions via proximity ligation assays

doi: 10.1016/j.xpro.2026.104361

Figure Lengend Snippet: Potential background noise of PLA dots (A) The amount of freeze-thaw cycles of the PLA kit influences the number of PLA dots. The more often the kit was thawed, the weaker the PLA will work, which also means that the background noise in the vector transfected cells will reduce. Red, PLA; blue, DAPI. Scale bar: 20 μm. (B) Dilution series of anti-StrepII antibody influences the amount of PLA dots, and can prevent increased background staining. Red, PLA; blue, DAPI. Scale bar: 10 μm.

Article Snippet: Monoclonal mouse anti-StrepII-tag Antibody (517) (PLA 1:400) , Novus Biologicals , Cat#NBP2-43735.

Techniques: Plasmid Preparation, Transfection, Staining