spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

Right arrow Help viewing high resolution images
Right arrow Return to article
(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 6


Fig. 6. The caveolin-1 scaffolding domain is required for interaction of EphB1 with caveolin-1 and for ERK activation by EphB1. (A,B) Expression of caveolin-1 in Cos-7 cells, Cos-7 cells transfected with wild-type caveolin-1 (Cav-wt), and CHO-EphB1 by immunoblotting (A) and by immunofluorescence (B1-B3, respectively). (B) The cells were fixed and stained with anti-caveolin-1 primary antibody followed by Alexa red-conjugated secondary antibody. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscopy, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Bars, 20 µm. (C) Cos-7 cells were transiently transfected with empty vector, wild-type (Cav-wt) or mutant caveolin-1 (Cav-mut) constructs, and additionally with wild-type EphB1 receptor: non-transfected Cos-7 cells (lane 1), empty vector (lanes 2-3), Cav-wt construct (lanes 4-5), and Cav-mut construct (lanes 6-7). Cells were co-transfected with wild-type EphB1 receptor (lanes 2-7). After 48 hours, the cells were incubated for 30 minutes with 1-2 µg/ml ephrinB2/Fc. Upper panel, co-immunoprecipitation of EphB1 receptor with anti-caveolin-1 antibody. The immunoprecipitates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with anti-HA or anti-caveolin-1, respectively. Lower panel, ephrinB2/Fc-induced ERK phosphorylation following transfection with the mentioned constructs was assessed using a phosphospecific antibody, then stripped blots were reprobed with anti-ERK antibody. (D) Cos-7 cells were transiently transfected with Cav-wt or Cav-mut constructs and additionally with wild-type or mutant EphB1 receptor: non-transfected Cos-7 cells (D1), Cos-7 cells transfected with wild-type EphB1 receptor and Cav-wt (D2), mutant EphB1 receptor and Cav-wt (D3), wild-type EphB1 receptor and Cav-mut (D4), and mutant EphB1 receptor and Cav-mut (D5). The cells were fixed and stained with anti-HA and anti-caveolin-1 primary antibodies followed by Alexa green- and red-conjugated secondary antibodies, respectively. Samples were processed as described under Materials and Methods. Images were acquired with a Nikon Eclipse E600 microscopy, connected to Nikon digital camera DXM1200, and processed with the Nikon AC-1 software version 2.11. Magnification, x1000. Results are representative of at least three independent experiments.





Right arrow Return to article