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Figure 3


Fig. 3. Expression of endogenous hCL species and RAMP mRNA in endothelial cells. (A) Expression of endogenous hCL species was analysed by immunoblotting. hDMVEC lysates were treated with endoglycosidase F (F, lane 2), endoglycosidase H (H, lane 3) or vehicle (-, lane 1) before SDS-PAGE under reducing conditions and immunoblotting with polyclonal anti-hCL antibody LN-1436. Arrowheads, deglycosylated (~37 kDa); open diamonds, core-glycosylated (~45 kDa); black diamonds, mature fully glycosylated (~55 kDa) forms of the receptor. The ~55 kDa hCL species are reduced to a ~37 kDa hCL band after endoglycosidase F treatment, but are resistant to endoglycosidase H. For loading controls, the membrane was reprobed with an antibody against ß-actin. The immunoblot is representative of two independent experiments. (B) Expression of CL and RAMP mRNAs in primary hDMVECs (lane 1) was analysed by RT-PCR. The set of primers for detection of ß-actin was used as a loading control. RNA sample from kidney (lane 2) served as a positive control. Numbers to the right indicate PCR fragment size. (For details of a full RT-PCR screen of all endothelial cell lines used in the present study, see supplementary material, Fig. S5.)





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