Fig. 5
uPARAP forms a complex with VEGFR-2 and VEGFR-3 and restricts VEGFR-2/VEGFR-3 heterodimerisation. In situ PLA in LECs (a, b, e–i) or PAECs (d) stimulated or not for 5 (T5) and 30 min (T30) with VEGF-C (a, b, e–g). a, b Interaction between uPARAP and VEGFR-2 (a) or VEGFR-3 (b). Results are those of one representative assay out of 3. Data represent mean (biological duplicates, 20 images analysed for quantification) ± SEM. Bars = 10 µm. c Immunoprecipitation (IP) of uPARAP at the indicated time point of VEGF-C stimulation. Western blots were revealed with an antibody (IB) against VEGFR-2, VEGFR-3 (upper panel) or uPARAP (lower panel). GAPDH was used as loading control. d In situ PLA detection of uPARAP interaction with VEGFR-2 or VEGFR-3 under basal conditions, in PAECs expressing VEGFR-2 (PAEC-VEGFR-2), VEGFR-3 (PAEC-VEGFR-3) or no VEGFR (PAEC). Bars = 10 µm. e, f In situ PLA detection of VEGFR-2/VEGFR-3 heterodimerisation in control LECs (Ctr), LECs deficient for uPARAP expression (siU1 and siU2) (e), or LECs overexpressing uPARAP (uPARAP cDNA) (f). g VEGFR-3 homodimerisation in LECs transfected with a VEGFR-3-Flag-tagged cDNA. Data correspond to the mean (biological triplicates, 60 images analysed for quantification, >100 cells analysed per condition) ± SEM (e–g). h In situ PLA detection of uPARAP/VEGFR-2 and uPARAP/VEGFR-3 complexes in LECs migrating in a VEGF-C gradient. Bars = 20 µm. i In situ PLA detection of VEGFR-2/VEGFR-3 heterodimers in control LECs (Ctr) and uPARAP silenced LECs (siU) migrating in a VEGF-C gradient. The left histogram corresponds to the mean number of dots per cell. The right histogram represents PLA signal localisation with respect to the nucleus (one representative experiment out of 3 with 25 cells analysed per condition in each experiment). Bars = 20 µm. Statistical analyses were performed using a non-parametric Mann–Whitney test. i Chi-square test was used. *P < 0.05, **P < 0.01, and ***P < 0.001
