Journal: Circulation: Cardiovascular Genetics

Article Title: The Role of CAV3 in Long–QT Syndrome

doi: 10.1161/circgenetics.113.000137

Figure Lengend Snippet: Figure 5. A, Western blot analysis of immunoprecipitated human embryonic kidney 293 protein extracts. Double-transfected HEK293 cells with KCNH2 and CAV3 were immunoprecipitated with anti-Kv11.1 antibodies and blotted for the presence of caveolin-3. Lane 2 shows that caveolin-3 coimmunoprecipitates with Kv11.1. Cells transfected with only 1 of the constructs (CAV3 or KCNH2) and cells transfected with empty plasmids were used as controls (lanes 5, 7, and 9). No caveolin-3 is detected in any of the control lanes, including the sample, where immunoprecipitation was carried out with normal goat serum polyclonal IgG (lane 3) instead of anti-Kv11.1 antibodies, thus confirming the specificity of the Kv11.1–caveolin-3 binding. B, Western blot analysis of immunoprecipitated Kv11.1 wild-type (WT) and mutant protein. The caveolin-3:p.T78M and the Kv11.1:p.I400N mutants were investigated in coimmunoprecipitation experiments and compared with WT. The interaction between caveolin-3 and Kv11.1 mutants is not disrupted in any combination as shown by the presence of the caveolin-3 band in all the immunoprecipitation lanes.

Article Snippet: The human KCNH2 and the CAV3 cDNA constructs were purchased from OriGene Technologies, Inc, (Rockville, MD).

Techniques: Western Blot, Immunoprecipitation, Transfection, Construct, Control, Binding Assay, Mutagenesis