Fig 1.
The bPHA specifically detects the proximal localization of BCRs on Ramos cell surface.
(A-D) Schematic presentation of bPHA. The proximity of protein A and protein B is converted to the proximity of plus and minus oligos. The oligos are then hybridized to Z-DNA, followed by preamplifier, amplifier, and finally, fluorescent label probes. (E) Diagram showing that changing a part of the Z-DNA allows the change of fluorescence of bPHA signal. (F) Schematic presentation of TD05 aptamer and the TD05− and TD05+ derivatives. (G) Flow cytometry results showing similar staining of Ramos cells with Cy5-labeled TD05, TD05+, and TD05−. Cells stained with Cy5-coupled unrelated aptamer functioned as negative control. (H) TD05+:TD05− bPHA signal measured by flow cytometry for Ramos cells treated with the indicated probes. (I) Confocal microscopic images of Ramos cells after bPHA and anti-IgM staining. (G-I) Data represent at least three independent experiments. (J) Schematic drawing explaining what could be measured by the TD05+:TD05− bPHA. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; Cy5, cyanine 5; HC, heavy chain; IgM, immunoglobulin M; LC, light chain; MFI, mean fluorescence intensity.
Fig 2.
The bPHA provides an excellent quantitative measurement of protein proximity in a large dynamic range.
(A) Schematic illustration showing that every GFP-μm expressed on Ramos cells surface have two GFPs in proximity that could be detected by Enh+:Enh− bPHA. (B) Flow cytometry results showing that the surface GFP-μm expression as assayed by Enh Cy5 staining is correlated with the GFP expression level. Gated for GFP+ cells. (C) Enh+:Enh− bPHA signal measured by flow cytometry for GFP− and GFP+ cells. (D) Flow cytometry results showing that the Enh+:Enh− bPHA signal is correlated with GFP signal. Gated for GFP+ cells. (E) Statistical analysis for data presented in (D). Raw data used for this plot are included in S1 Data. (F) Enh:Enh PLA signal measured by flow cytometry for GFP− and GFP+ cells. (G) Flow cytometry results showing that the Enh:Enh PLA signal is not correlated with GFP signal. Gated for GFP+ cells. (H) Statistical analysis for data presented in (G). Raw data used for this plot are included in S2 Data. Data are representative of a minimum of three independent experiments. bPHA, branched proximity hybridization assay; Cy5, cyanine 5; Enh, Enhancer; GFP, green fluorescent protein; PLA, proximity ligation assay.
Fig 3.
The bPHA works in a mixed cell population.
(A) Schematic diagram showing the route map for generating GFP-μm-expressing IgM or IgD KO cells from WT Ramos B cells by CRSPR/Cas9 method. (B) Flow cytometry results showing that the subpopulations of the mixed cells can be identified by gating for the expression of GFP and anti-IgD staining. (C and D) TD05+:TD05− (C) or Enh+:Enh− (D) bPHA results were measured by flow cytometry and analyzed by the gating strategy shown in (B). Data represent three independent experiments. bPHA, branched proximity hybridization assay; Enh, Enhancer; GFP, Green fluorescent protein; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out; PE, phycoerythrin; WT, wild-type.
Fig 4.
The bPHA confirms the rearrangement of BCR upon stimulation.
(A) TD05+:TD05−, TD05+:Enh−, and Enh+:Enh− bPHA signals were measured by flow cytometry for resting and stimulated IgM-KO GFP-μm-expressing Ramos cells. The stimulated cells without the corresponding target binding probes served as control. Data represent four independent experiments. (B) Schematic diagrams showing that on the surface of IgM-KO GFP-μm-expressing Ramos cells surface, upon stimulation, IgD-BCR and GFP-μm mix together, producing positive TD05+:Enh− bPHA signal. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; Enh, Enhancer; IgD, immunoglobulin D; IgM, immunoglobulin M; KO, knock-out.
Fig 5.
The bPHA uncovers class-specific kinetics of Syk recruitment to BCR.
(A) Schematic diagrams showing that upon stimulation, Syk can be recruited to CD79a. The proximity between CD79a and Syk can be measured by bPHA using oligo-coupled anti-CD79a and anti-Syk antibodies. (B) Anti-CD79a+:anti-Syk− bPHA signals were measured by flow cytometry for resting and anti-IgD- or anti-IgM-stimulated splenic B cells. Cells without the corresponding target binding probes served as controls for both the resting and stimulated cells. Data represent three independent experiments. BCR, B cell antigen receptor; bPHA, branched proximity hybridization assay; IgD, immunoglobulin D; IgM, immunoglobulin M; Syk, spleen tyrosine kinase.