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. 2010 Mar 11;29(8):1377–1388. doi: 10.1038/emboj.2010.30

Figure 2.

Figure 2

In situ PLA detection of VEGFR2/-3 heterodimers in intact HSaVECs. (A) Schematic outline of the in situ PLA strategy showing: (i) dimerized receptors (VEGFR2 in blue and VEGFR3 in grey) reacting with primary antibodies; (ii) close proximity of oligonucleotide-ligated secondary antibodies allows a rolling-circle amplification (RCA); (iii) detection of the RCA product by a fluorescently labelled probe. (B) Detection of heterodimers (in red) in HSaVECs treated with vehicle (–), VEGFA or VEGFC for 8 min on cells labelled with FITC-conjugated phalloidin (green). Inset in the VEGFC panel shows high magnification to clearly visualize the PLA spots representing heterodimers. Scale bar=10 μm. (C) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with vehicle (–), VEGFA (A) or VEGFC (C) in cells preincubated or not with neutralizing antibodies blocking ligand binding to VEGFR2 or VEGFR3. n=6. (D) Quantification of VEGFR2/-3 heterodimers in HSaVECs treated with different human VEGF isoforms (VEGFA121, 145, 165 or 189) or VEGFC for 8 min. n=6. (E) Quantification of VEGFR2/-3 heterodimers in response to VEGFA, VEGFC, VEGFD or PDGFB. Growth factors are indicated as A (VEGFA), C (VEGFC), D (VEGFD) and P (PDGFB). n=6. Note that a different batch of PLA probes was used in this analysis compared with other panels in the figure (see Materials and methods). (F) Turnover of VEGFR2/-3 heterodimers in HSaVECs. Cells were treated with VEGFC for different time periods from 10 min to 24 h and samples were processed for detection of in situ PLA signals. n=6. Asterisks in panels C–F indicate the degree of significance (**P<0.01, ***P<0.001).