TI - Results Generation of stable clones expressing wild-type and mutant PKD2 in HEK293 cells . AB - To test the role of PKD2 in renal cell proliferation and specifically on the p21/Cdk2 pathway , we generated a series of HEK293 cell lines with stable expression of hemaglutinin ( HA ) -tagged wild-type human PKD2 ( WT PKD2 ) , HA-tagged mutant PKD2 ( R742X PKD2 ) and a selectable marker ( Vector ) . The R742X PKD2 encodes for a truncated PC-2 lacking the polycystin-1 ( PC-1 ) interacting region at the carboxy terminal of the protein . R742X , is a disease-causing PC-2 mutant firstly identified in a Greek-Cypriot family with Polycystic Kidney Disease type 2 [26-28] . Three individual clones were isolated from each transfectant and used for further experimentation . Immunoblotting of whole cell lysates from the selected clones with an HA antibody , showed good expression of HA-tagged WT PKD2 and HA-tagged R742X PKD2 (Figure 1A) . The same lysates were immunoblotted with anti-PC-2 antibody to demonstrate that we indeed have PC-2 overexpression in these clones . As seen in figure 1A , endogenous PC-2 is barely detectable by Western blot analysis in vector-only and R742X PKD2 transfectants . The lower molecular weight band detected most likely represents a non-specific band detected with the anti-PC-2 antibody , since it is detected on vector-only transfectants and untransfected cells ( Figure 1A and data not shown ) . We used these tools to test the effect of wild-type and mutant PC-2 expression on the JAK2/STAT-1/p21/Cdk2 pathway , as it was previously implicated in its regulation by showing that overexpression of wild type PKD1 activates JAK2 kinase , which in turn PHOSphorylates STAT-1 [13] . Lysates from synchronized clones were immunoblotted with an anti-phosphorylaTED STAT-1 antibody , which detects the expression of serine PHOSphorylated STAT-1 , and an anti-p21 to detect endogenous p21 expression . As shown in figure 1A , p21 levels and STAT-1 PHOSphorylation were unaffected by wild-type or mutant PKD2 expression . Equal loading was confirmed by re-probing the same membrane with anti-beta-tubulin . Similarly , endogenous Cdk2 activity was equivalent among the different clones as judged by the kinase assay performed on Cdk2 immunoprecipitates from two selected clones of each transfectant . Western blot analysis demonstrated that similar amount of Cdk2 was precipitated from each clone (Figure 1B) . Cell cycle analysis performed by propidium iodide ( PI ) staining revealed that expression of wild-type or mutant PC-2 does not alter the cell cycle profile of these cells ( Figure 2 ) . Furthermore , proliferating cell nuclear antigen ( PCNA ) levels were equal among the different clones (Figure 1A) . Collectively , the results suggest that expression of wild-type and mutant PKD2 has no effect on the proliferation of HEK293 cells . To determine whether mislocalization of exogenous WT and R742X PC-2 is responsible for their inability to regulate cellular proliferation , we compared the sub-cellular localization of HA-tagged WT or R742X PC-2 with endogenous PC-2 by immunofluoresence . Both HA-tagged WT and R742X PC-2 were detected at the same subcellular compartments ( endoplasmic reticulum and plasma membrane ) as the endogenous PC-2 ( data not shown ) . ER-localized PC-2 is known to function as a Ca2+ -activated intracellular Ca2+ release channel while plasma membrane-associated PC-2 is believed to function as a nonselective cation channel [29-31] . Previous work has demonstrated that PKD2 overexpression augmented the amplitude of whole cell currents in renal epithelial cells [20] . To test the effectiveness of the expressed WT PKD2 in HEK293 cells we performed whole cell current measurements in vector-only , WT PKD2 and R742X PKD2 clones . Functional expression of transfected wild type PKD2 in HEK cells has been shown [32] . Figure 3 shows that stable expression of wild type PKD2 in HEK cells resulted in a significant increase in the current amplitude of whole cell inward currents recorded either in normal extracellular tyrode solution or symmetrical K+ ( Figure 3 ) . Outward currents were larger in WT PKD2 expressing cells compared to untransfected , mock-transfected , or R742X PKD2 -transfected cells in symmetrical K+ . PKD2 -mediated K+ currents were larger compared to Na+/Ca2+ currents as was expected for PKD2 which shows higher permeability to K+ compared to Na+ or Ca2+ [16 , 20] . Overexpression of R742X PKD2 did not have a significant effect on whole cell inward or outward currents in HEK293 . Collectively , the electrophysiology data show that transfection of wild type PKD2 resulted in functional expression in HEK293 cells . However , PKD2 has no effect on the STAT-1/p21/Cdk2 pathway or on the proliferation status of these cells .