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pmic7318-sup-0001-FigureS1.jpg318KFigure S1: Western blots were performed using Min6 membrane preparations. RSUR1, CSUR1 and W62 antibodies were used to detect endogenous SUR1 and Kir6.2 proteins. Interestingly, a double band was detected by the CSUR1 antibody with the lower of the two bands presumably reflecting a non-glycosylated form of the protein [1].
pmic7318-sup-0002-FigureS2.jpg510KFigure S2: Characterization of solubilization conditions and antibodies used. A: Following preparation of Min6 membranes, samples were solubilized Triton X-100, NP40, digitonin (DG), n-octylglucoside (OG), dodecylmaltoside (DM) or Chaps in different concentrations (0.1 to 1 % v/v) before subjected to Western blotting with the RSUR1 antibody. B: Immunoprecipitations were performed using W62b and M19 (left panel) or C62 and G16 (middle panel) antibodies and rat ventricular myocyte membranes with the amine-reactive resin system. Also immunoprecipitations were performed using the RSUR1 antibody and Min6 membranes with Protein A/G Dynabeads (right panel). The presence of Kir6.2 in the immunoprecipitates was detected by C62 (left panel), W62b (middle panel) or G16 antibody (right panel) as a 37 kD band (long arrows). Immunoprecipitations with unrelated IgG were used as negative controls. Non specific antibody bands are detected when the Protein A/G beads are used for the immunoprecipitation (marked as IgG, right panel).
pmic7318-sup-0003-FigureS3.pdf130KFigure S3: FDR calculation of KATP channel interacting proteins. In most cases proteins were detected only in immunoprecipitates with anti-channel antibodies and not in control IgG immunoprecipitates. For cases in which a protein was detected in both channel and control immunoprecipitates, we calculated the probability that enrichment was significant as follows: (A) The intensity ratios for all proteins with no missing data (“3+3”; found in 3 out of 3 technical replicates for channel IP and 3 out of 3 technical replicates for IgG negative control immunoprecipitations) were plotted and used to estimate a simulated coefficient of variation of 0.5. The X-axis represents the log2 ratio of the channel immunoprecipitation over the IgG immunoprecipitation. The Y-axis represents the number of protein hits. (B) The ratio distribution due to random intensity variation was simulated for cases when there were missing data. The X-axis represents the log2 ratio of channel immunoprecipitation over IgG immunoprecipitation. The Y-axis represents the number of normalized protein hits. (C) The simulated ratio distributions were used to calculate the false discovery rate (FDR) as a function of the ratio. The graph shows the FDR as a function of log2 ratio (channel immunoprecipitation over IgG immunoprecipitation) for cases where there is no missing data (“3+3”) and cases with missing data (“3+2”, “3+1”, etc.) and is normalized to have a FDR of 1 at a ratio of negative infinity.
pmic7318-sup-0004-TableS1.pdf295KTable S1.
pmic7318-sup-0005-TableS2.docx39KTable S2: Functional enrichment of the proteins identified in Table S1.

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