Figure 1
Figure 1. Analysis of AKAP9 350/450 expression in human ECs and characterization of cell cycle in AKAP9-depleted cells. (A) Left panel, Western blotting (WB) of AKAP9 in HUVEC lysate and 293 cells transfected with Yotiao (293-Yotiao). A single band at the predicted molecular weight for AKAP350/450 long isoform(s) (arrow) was observed in HUVECs. The lower 250-kD immunoreactive band in HUVECs did not migrate at the position of recombinant Yotiao in 293 cells (arrowhead). Right panel, qRT-PCR of HUVECs using primers that recognize the long isoforms (350/450 isoforms) or Yotiao was undertaken and their relative abundance (percentage of total AKAP9) was calculated. n = 4. (B) Confocal analysis of HUVECs stained with antibodies to the indicated proteins and DAPI (nucleus stain) followed by overlay of the images. AKAP9 (Akp9) codistributed with γ-tubulin, a centrosomal marker (top panel, arrow), and colocalized with GM-130, a cis-Golgi marker that is perinuclear and excludes the centrosome (bottom panel, arrow). Bar = 10 μm. (C) Tissue sections of human skin were stained with rabbit anti-Akp9, anti–PECAM-1 or rabbit IgG. AKAP9 was present in the large and small vessels (arrow) of the skin in a pattern coincident with an endothelial cell marker, PECAM-1, and in the epithelium. No specific staining was observed with IgG. (D) HUVECs were transfected with AKAP9 (Akp9) or Control (Con) siRNA duplexes (si), and evaluated 48 hours later for AKAP9 expression. Left panel, AKAP9 protein level was analyzed by Western blotting. The lower 250-kDs protein remained upon AKAP9 silencing, suggesting that it is a nonspecific, immunoreactive band. Middle panel, AKAP9 message was quantitated by qRT-PCR using oligonucleotides for long isoforms 350/450 kDa. Right panel, AKAP9 protein was assessed by immunofluorescence staining. (E) Representative cell-cycle profiles by fluorescence-activated cell sorting analysis of DNA content. Top left panel, AKAP9 silencing does not alter the distribution of cells in the different phases of the cell-cycle compared with control siRNA cells. Bottom left panel, an average of results from 3 independent experiments are given. Right panels, phase contrast images of control and AKAP9-silenced monolayers revealed an intact monolayer and cells that were morphologically similar in both cultures.

Analysis of AKAP9 350/450 expression in human ECs and characterization of cell cycle in AKAP9-depleted cells. (A) Left panel, Western blotting (WB) of AKAP9 in HUVEC lysate and 293 cells transfected with Yotiao (293-Yotiao). A single band at the predicted molecular weight for AKAP350/450 long isoform(s) (arrow) was observed in HUVECs. The lower 250-kD immunoreactive band in HUVECs did not migrate at the position of recombinant Yotiao in 293 cells (arrowhead). Right panel, qRT-PCR of HUVECs using primers that recognize the long isoforms (350/450 isoforms) or Yotiao was undertaken and their relative abundance (percentage of total AKAP9) was calculated. n = 4. (B) Confocal analysis of HUVECs stained with antibodies to the indicated proteins and DAPI (nucleus stain) followed by overlay of the images. AKAP9 (Akp9) codistributed with γ-tubulin, a centrosomal marker (top panel, arrow), and colocalized with GM-130, a cis-Golgi marker that is perinuclear and excludes the centrosome (bottom panel, arrow). Bar = 10 μm. (C) Tissue sections of human skin were stained with rabbit anti-Akp9, anti–PECAM-1 or rabbit IgG. AKAP9 was present in the large and small vessels (arrow) of the skin in a pattern coincident with an endothelial cell marker, PECAM-1, and in the epithelium. No specific staining was observed with IgG. (D) HUVECs were transfected with AKAP9 (Akp9) or Control (Con) siRNA duplexes (si), and evaluated 48 hours later for AKAP9 expression. Left panel, AKAP9 protein level was analyzed by Western blotting. The lower 250-kDs protein remained upon AKAP9 silencing, suggesting that it is a nonspecific, immunoreactive band. Middle panel, AKAP9 message was quantitated by qRT-PCR using oligonucleotides for long isoforms 350/450 kDa. Right panel, AKAP9 protein was assessed by immunofluorescence staining. (E) Representative cell-cycle profiles by fluorescence-activated cell sorting analysis of DNA content. Top left panel, AKAP9 silencing does not alter the distribution of cells in the different phases of the cell-cycle compared with control siRNA cells. Bottom left panel, an average of results from 3 independent experiments are given. Right panels, phase contrast images of control and AKAP9-silenced monolayers revealed an intact monolayer and cells that were morphologically similar in both cultures.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal