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. 2015 Jun 19;35(14):2495–2502. doi: 10.1128/MCB.01453-14

FIG 4.

FIG 4

Rap1 recruits ArhGAP29 to the plasma membrane through Radil. (A) In the upper panel, GFP-ArhGAP29 expressing HUVECs were treated with control siRNA (siC), siRNA targeting Radil and Rasip1 (siRadil + siRasip1), and Radil (siRadil) or Rasip1 (siRasip1). The cells were grown to confluence and treated with 007-AM for 15 min. The boxed areas of cell-cell contacts are enlarged in the insets. In the lower panel, the left graph shows the relative intensity profiles of fluorescent signal intensities along the line scans depicted in the boxed area. The right graph shows the knockdown efficiency as assessed by Q-PCR. (B) Live imaging of YFP-ArhGAP29 in HEK293T cells transfected with HA-Rap1A(V12) and YFP-ArhGAP29 upon cotransfection with either V5-Radil and HA-Rasip1 (first panel), empty vector only (second panel), HA-Rasip1 supplemented with empty vector (third panel), or V5-Radil supplemented with empty vector (fourth panel). (C) Live imaging of YFP-ArhGAP29 in HEK293T cells transfected with HA-Rap1A(V12) and YFP-ArhGAP29 upon cotransfection with either V5-Radil and HA-Rasip1 (first panel), V5-Radil K79A (V5-Radil KA) and HA-Rasip1 (second panel), or V5-Radil and HA-Rasip1 K163A (HA-Rasip1 KA) (third panel). The experiments were repeated at least three times, and representative images were chosen.