Table 1.
Interpreting the size of a correlation coefficient.
Fig 1.
Confocal immunofluorescence microscopy revealed differential VEGFR expression.
(A–H) A representative image of VEGFR1 co-localized with VEGFR2 in PC12 neuronal and mouse venous endothelial cells. Receptor expression was observed in both, endothelial and neuronal cells with distribution being more nuclear in neuronal cells as compared to endothelial cells. (A and E) VEGFR2 immunostaining revealed distinct receptor expression in endothelial and neuronal cells. (B and F) VEGFR1 surface immunostaining revealed punctate receptor expression in endothelial and neuronal cells. (C and G) DAPI nuclear staining. (D and H) Merging of the channels revealed co-expression of both receptors VEGFR1 and VEGFR2 in neuronal and endothelial cells. Scale bar, 10μm.
Fig 2.
VEGFR1 and VEGFR2 protein expression was different in neuronal versus endothelial cells.
(A and C) Representative images of immunoblots probed with anti-VEGFR1 (Panel A) or anti-VEGFR2 antibody (Panel C) showed differences in protein levels between endothelial and neuronal cells as determined by band intensity. HUVECs were used as positive controls (B and D) Fold increase in VEGFR1 (Panel B) and VEGFR2 (Panel D) expression was observed in both mouse venous (MVEC; VEGFR1-Red Striped Bar; VEGFR2-Blue Striped Bar) and aortic (MAEC; VEGFR1-Red Dotted bar; VEGFR2-Blue Dotted Bar) endothelial cells compared to PC12 neuronal cells (VEGFR1-Red Solid Bar; VEGFR2-Blue Solid Bar). GAPDH was used as a loading control; Fold change values are Mean ± SEM; n = 3; * P < 0.05 for relative VEGFR1 and VEGFR2 levels in endothelial vs neuronal cells (E) Relative VEGFR2 / VEGFR1 ratios were higher in both neuronal and endothelial cells (PC12-Violet Solid Bar; MVEC-violet striped bar; MAEC-violet dotted bar). Relative receptor ratios are Mean ± SEM; n = 3; * P < 0.05 for VEGFR2 / VEGFR1 ratio in endothelial cells vs neuronal cells (F) Relative VEGFR1 / VEGFR2 ratios were lower in both neuronal and endothelial cells (PC12-Green Solid Bar; MVEC-Green striped bar; MAEC-Green dotted bar). Relative receptor ratios are Mean ± SEM; n = 3; * P < 0.05 for VEGFR1 / VEGFR2 ratio in endothelial vs neuronal cells.
Fig 3.
Immunoprecipitation / pulldown assays confirm presence of VEGFR1-R2 heterodimers.
(A) Immunoblot showing VEGFR2 protein bands in PC12 neuronal and MVEC cell lysates immunoprecipitated with anti-VEGFR1 antibody (B) Immunoblot showing VEGFR2 protein bands in MAEC and HUVEC cell lysates immunoprecipitated with VEGFR1 antibody (C) Immunoblot from (Panel A), washed and reprobed with anti-VEGFR1 antibody showed VEGFR1 protein bands (D) Immunoblot from (Panel B) washed and reprobed with anti-VEGFR1 antibody confirming VEGFR1 pulldown. Cell lysates from PC12 neuronal cells as well as MAEC, MVEC and HUVECs when immunoprecipitated with anti-VEGFR1 antibody demonstrated the existence of VEGFR1-R2 heterodimers when probed with anti-VEGFR2 antibody (E, F) Immunoblots showing GAPDH, used as a loading control (G) Immunoblot showing VEGFR2 protein bands in tissue lysates immunoprecipitated with anti-VEGFR1 antibody confirm VEGFR1-R2 heterodimers. (H) VEGFR1 protein bands in tissue lysates confirmed VEGFR1 pulldown (I) GAPDH was used as a loading control.
Fig 4.
Visualization of VEGFR1-R2 heterodimers by TIRF microscopy.
(A-B) MVEC showed presence of VEGFR1 and VEGFR2 surface receptors observed as red and green fluorescent signals respectively (C-D) Merged image and linear graph showing positive VEGFR1-R2 colocalization (E-F) MAEC exhibiting VEGFR1 and VEGFR2 immunostaining (G-H) Merged image and receptor colocalization graph confirmed VEGFR1-R2 heterodimer presence (I-J) Expression of VEGFR1 and VEGFR2 surface receptors in trigeminal ganglion neuronal cells (K-L) Merged image depicting heterodimer presence and colocalization graph confirming the same (M-N) PC12 neuronal cells exhibited VEGFR1 (fluorescent red punctate staining) and VEGFR2 (fluorescent green puncta) surface receptors (O-P) VEGFR1-R2 heterodimers visualized as yellowish-orange fluorescence and graph showing colocalization between the 2 channels (Q) Pearson Correlation Coefficient values were high positive for neuronal and endothelial cells indicating strong colocalization and evidence of receptor heterodimers.
Fig 5.
Duolink PLA staining reveals abundant VEGFR2-R2 homodimers and R1-R2 heterodimers.
(A-C) Negative controls (or no antibody controls) in which neuronal and endothelial cells lacked primary antibody treatment show absence of red PLA staining. (D-F) Low levels of VEGFR1-R1 homodimers were found in PC12, TG neuronal and MVECs (G-I) R1-R2 heterodimers were visualized as red punctate staining in both types of neuronal cells as well as MVECs (J-L) Highest PLA staining corresponding to VEGFR2-R2 homodimers was observed in MVECs as compared to PC12 and TG neuronal cells (M) Bar plot based on Mean PLA Fluorescence intensity per cell showed that VEGFR2-R2 homodimers were most abundant in endothelial and neuronal cells *P < 0.05 between dimers in PC12 cells; + P < 0.05 between dimers in MAEC; # P < 0.05 between dimers in MVEC. (N) Vertical stacked bar chart based on ratio of VEGFR dimer distribution to total number of dimers showed that neuronal cells had higher expression of VEGFR1-R2 heterodimers as compared to that in endothelial cells. All data are shown as Mean ± SEM (n = 3); * P < 0.05 vs R1-R1 homodimers.