Alx 215 049 r100

Cells were lysed 48 h post-transfection using buffer containing (in mM): 150 Tris-NaCl, 25 Tris HCl, 10 Na-EGTA, 20 Na-EDTA, 5 glucose, and supplemented with 1% Triton X-100, 50 µg/ml 1,10 phenathroline, 0.7 µg/ml pepstatin A, 1.56 µg/ml benzamidine and 1x Complete Minitab (Roche Applied Science). Solution was sonicated, incubated on ice for 30 min, and centrifuged for 10 min at 13,000 rpm at 4°C. Protein concentration of supernatant was assessed using a BIORAD protein Assay. 30 µg of protein per lane was electrophoresed on a 7.5% SDS-polyacrylamide gel and then transferred onto PVDF membranes. Membranes were blocked and incubated overnight with 1:2000 anti β-actin (ab8226, abCam), and either anti-hERG (CT) (pan) (ALX-215-049-R100, Enzo Life Sciences) or anti-Ribosomal Protein L13A (C-11) (sc-390131, Santa Cruz Biotechnology). Membranes were washed and then incubated with 1:1000 dilutions of secondary antibody Alexa Fluor® 647 Goat Anti-Rabbit IgG A-21245 (Life Technologies) for 1 hour and imaged using a Chemidoc-MP Imaging System (BIORAD). Individual values were calculated from the mean of three parallel experiments completed on the same day and under identical conditions (i.e. n = 1). Protein values were then paired with experiments completed on the same day to increase statistical power.

Purified protein was co-immunoprecipitated as previously described (Phartiyal et al., 2007). Briefly, transiently HEK293 whole cell lysates (500μg) were precleared with 30µl protein G sepharose beads for 1 hour at 4 °C. Following a 1 minute at 10,000 g, centrifugation at 4 °C to remove beads, 5 µl mouse anti-myc (Abcam) was added to the supernatant and incubated for 16 hours at 4 °C, rotating. For biotinylation preps we replaced anti-myc with the anti-hERG (ALX-215-049-R100, Enzo Life Sciences). Protein G sepharose beads were then added and incubated for an additional 2h. Immunoprecipitates were washed three times in 0.5 ml lysis buffer, and eluted into 30µl Laemmeli sample buffer (25 mM Tris-HCl, pH 6.8, 2% sodium dodecysulfate, 10% glycerol, 0.2 M DL Dithiothreitol) and subjected to a western blot probed with rabbit-pan hERG antibody (Enzo Life Sciences).