Figure 3.

Electrophoretic mobility shift assay and subsequent protein sequencing (EMSA-PSeq). The dsDNA probes for SCNN1B SNP rs152731 allele C, SCNN1B SNP rs152731 allele T and the NFkappaB-P65 consensus motif¹⁶ were incubated with T84 nuclear extract. After non-denaturing 7% polyacrylamide electrophoresis, the samples were transferred by electrotransfer in a denaturing, SDS-containing buffer onto a membrane assembly whereby the gel was covered with an uncharged nylon membrane (Hybond C, Amersham), followed by two charged nylon membranes (Hybond N+, Amersham). Unbound oligonucleotides were transferred through the uncharged membranes and could be visualized on the charged nylon membranes (SupplFig4, SupplFig5). Probes derived from rs152731 gave rise to a high molecular weight complex I which differs from complex II generated with an NFkappaB-P65 consensus motif. P65, STAT3 and STAT6 were identified in the high molecular weight complex II, whereby for this experiment, the position corresponding to the visualized signal was excised from the second gel half loaded identically without electroblotting and subjected to protein mass spectrometry.