Journal: Pacing and clinical electrophysiology : PACE

Article Title: Partially dominant mutant channel defect corresponding with intermediate LQT2 phenotype.

doi: 10.1111/j.1540-8159.2011.03222.x

Figure Lengend Snippet: Figure 4. Cell surface expression of G816V HERG compared to WT HERG. (A) Immunoblot of cell surface proteins that were isolated by biotin labeling and pull-down from transiently transfected HEK cells. Samples were separated by 7.5% linear SDS-PAGE. Calnexin was the negative control for surface labeling and cadherin was the positive control for surface labeling in the lower panels. Western blot was performed with anti-HERG, anti-calnexin, and anti-cadherin antibodies. (B) Summary data for part A. Densitometry analysis of the surface expression of HERG proteins was quantified as the amount of surface HERG divided by the total cellular HERG and also normalized for streptavidin pull-down of biotinylated protein (normalization calculated using cadherin and calnexin controls). WT HERG is normalized to 1.0. n = 4, *P < 0.05. (C) Confocal immunofluorescence micrographs of HEK 293 cells transfected with either WT HERG or G816V HERG and counter-stained with anti-cadherin antibody to indicate the cell membrane. Pearson correlation of HERG and cadherin colocalization is 0.571 ± 0.025 for WT HERG and 0.332 ± 0.029 for G816V, P < 0.001. n = 15, scale bar = 10 μm, DIC = differential interference contrast. (D) Confocal immunofluorescence micrographs of HEK 293 cells transfected with either HERG-WT or HERG-G816V and counter- stained with anti-calnexin antibody to indicate the ER, an intracellular compartment. Pearson correlation of HERG and calnexin colocalization is 0.831 ± 0.026 for WT HERG and 0.916 ± 0.006 for G816V, P < 0.01. n = 13, scale bar = 10 μm.

Article Snippet: Loading control antibodies used were pan-cadherin (Abcam), calnexin (Santa Cruz Biotechnology), Na-K-ATPase (Upstate, Millipore, Billerica, MA, USA), and tubulin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Isolation, Labeling, Transfection, SDS Page, Negative Control, Positive Control, Staining, Membrane

Journal: Pacing and clinical electrophysiology : PACE

Article Title: Partially dominant mutant channel defect corresponding with intermediate LQT2 phenotype.

doi: 10.1111/j.1540-8159.2011.03222.x

Figure Lengend Snippet: Figure 5. Interaction of WT HERG and G816V HERG subunits. (A) Co-immunoprecipitation and subsequent Western blot from transiently transfected HEK 293 cells shows 3× FLAG-tagged WT HERG detected from a pull-down of myc-tagged G816V HERG in three different mixed combinations with 100% WT HERG and 100% G816V HERG as controls, n = 3. Lower panel shows the reverse experiment where myc-tagged G816V HERG was detected in a pull-down of 3× FLAG-tagged WT HERG from the same combinations, n = 3. (B) Immuno-blots of cell surface biotinylation of 3× FLAG-tagged WT HERG and myc-tagged G816V HERG alone or in a 50/50 mix. Proteins were separated by 7.5% SDS-PAGE. Top panel shows a blot with anti-FLAG antibody with cadherin and calnexin as controls. Lower panel shows a blot with anti-Myc antibody with cadherin and calnexin as controls, n = 3.

Article Snippet: Loading control antibodies used were pan-cadherin (Abcam), calnexin (Santa Cruz Biotechnology), Na-K-ATPase (Upstate, Millipore, Billerica, MA, USA), and tubulin (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Immunoprecipitation, Western Blot, Transfection, SDS Page