Figure 2.

Rap1A and Rap1B exhibit differences in subcellular localization; Rap1A is more prominent at cell junctions, co-localizing with VE-cadherin and co-immunoprecipitating with AF6. (A) GFP-tagged Rap1A or Rap1B were exogenously expressed in HUVEC and localization was visualized by fluorescence microscopy. Rap1A isoform is concentrated at cell-cell junctions; Rap1B has both perinuclear and weaker junctional fluorescence. Scale bar = 100 µm. (B) Localization of GFP-Rap1A (left column) or GFP-Rap1B (right column) and co-staining for VE-cadherin. Boxed areas in each VE-cadherin image have been enlarged in the merged parts, identifying Rap1 isoform localization in green and VE-cadherin localization in red. Rap1A more strongly colocalizes with VE-cadherin in areas of cell-cell contact, as indicated by yellow/orange coloring in the merged part (left). Scale bar = 50 µm. (C) Quantification of GFP-Rap1A vs. Rap1B junctional pixel intensity relative to total cellular GFP fluorescence. VE-cadherin was used as a marker for cell-cell junctions. Images were analyzed using ImageJ as described in Methods. The junctional proportion of Rap1A is significantly higher than Rap1B. Graph represents average junctional intensity of >10 cells per field (n = 9 fields) from 3 independent experiments *p < 0.01. (D) GFP-tagged proteins (GFP-CA-Rap1A, CA-1B or GFP alone) were expressed in HUVEC , followed by immunoprecipitation with anti-GFP antibodies. Lysates and IP samples were blotted with indicated antibodies. The junctional protein AF-6 more strongly co-immunoprecipitates with GFP-Rap1A. Equivalent expression level of GFP-Rap1A and 1B, was confirmed; endogenous Rap and AF-6 protein present in total cell lysates indicate equal loading.