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Fig. 1. Transiently expressed and endogenous hCL species. The human endothelial CL cDNA was cloned into the pcDNA 3.1 expression vector and termed hCLpcDNA. To examine the functionality, the resulting vector was transiently cotransfected with RAMPs into HEK293T cells and the intracellular cAMP concentrations were measured. The intact HEK293T cells lacked functional AM and CGRP receptors, because they showed little cAMP response to agonist stimulation. By contrast, in HEK293T cells cotransfected with hCL-RAMP, the receptors were fully functional in intracellular cAMP production depending on the ligand specificity of the RAMPs (data not shown). (A) Total cell or (B) tissue lysates were analysed by SDS-PAGE under reducing conditions, and immunoblots were probed using polyclonal anti-hCL antibody LN-1436. (A) The antibody specifically recognises hCL in HEK293T cells transfected with hCLpcDNA alone or together with RAMP1 or RAMP2 (CL+RAMP1 or CL+RAMP2; functional CGRP and AM receptors, respectively). HEK293T cells non-transfected (MOCK) or transfected with empty pcDNA vector (pcDNA) or RAMP1 or RAMP2 alone do not express hCL. The 
40-45 kDa hCL species (open diamonds; core-glycosylated receptor) are present in HEK293T cells transfected with hCLpcDNA only. The 55 kDa hCL species (black diamonds; mature fully glycosylated receptor) is only produced when the receptor is co-expressed with RAMPs. (For details of deglycosylation experiments confirming the origin of hCL species, see supplementary material, Fig. S2.) (B) The antibody also recognises endogenous hCL species expressed in tissues. Arrowheads, deglycosylated (37 kDa) form of the receptor. The immunoblots are representative of two independent experiments. For loading controls, the membrane was re-probed with an antibody against ß-actin.
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