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Fig. 2 | BMC Biology

Fig. 2

From: A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

Fig. 2

RNb and GNb fusion proteins bind to their respective tagged proteins in live cells. a Schematic of the RNb-GFP fusion binding to RFP. b HeLa cells expressing RNb-GFP with RFP-tagged markers for the ER surface (mCh-Sec61β), the mitochondrial surface (TOM20-mCh), the nucleus (H2B-mCh), or the surface of lysosomes (TPC2-mRFP). Cells were imaged in HBS using epifluorescence microscopy (cells expressing H2B-mCh) or TIRFM (other cells). Yellow boxes indicate regions enlarged in the subsequent panels. Colocalization values (Pearson’s coefficient, r) were mCh-Sec61β (r = 0.93 ± 0.09, n = 10 cells), TOM20-mCh (r = 0.94 ± 0.09, n = 10 cells), H2B-mCh (r = 0.97 ± 0.06, n = 10 cells), and TPC2-mRFP (r = 0.78 ± 0.09, n = 5 cells). c Schematic of the GNb-mCh fusion binding to GFP. d HeLa cells co-expressing GNb-mCh with GFP-tagged markers for the ER surface (GFP-ERcyt), the mitochondrial surface (TOM20-GFP), and the nucleus (H2B-GFP), or an mTurquoise2-tagged ER surface marker (mTurq-ERcyt). Cells were imaged using epifluorescence microscopy (cells expressing H2B-GFP) or TIRFM (other cells). Yellow boxes indicate regions enlarged in the subsequent panels. Colocalization values were GFP-ERcyt (r = 0.92 ± 0.08, n = 8 cells), TOM20-GFP (r = 0.87 ± 0.05, n = 7 cells), H2B-GFP (r = 0.94 ± 0.07, n = 6 cells), and mTurq-ERcyt (r = 0.97 ± 0.03, n = 7 cells). Scale bars 10 μm (main images) or 2.5 μm (enlargements)

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