Fig 2: The R507W substitution does not inhibit association of MT-A6 with WT-A6. The principal objective of this experiment was to determine if the p.R507W mutation affected the ability of the resultant mutant to associate with wild-type (WT) Slc26a6 and form the prototypic Slc26a6 multimeric complex. By using different epitope tags for the potential binding partners, we used anti-myc tag (WT) primary immunoprecipitation to assess association with HA-tagged protomers (WT +MT) by monitoring levels of coimmunoprecipitation. OKP cells were cotransfected (Co-Tx) as described for figure 3, solubilised with 1% digitonin and subjected to immunoprecipitation with a mouse anti-myc monoclonal antibody (Thermo Fisher R950-25; 5 µg IgG/mL lysate). Panel A: western analysis of myc-tagged WT Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel B: western analysis of HA-tagged Slc26a6 expression in total cell lysates isolated from each cotransfection. Panel C: western analysis of primary immunoprecipitated myc-tagged WT Slc26a6 from each cotransfection. Panel D: western analysis of coimmunoprecipitated HA-tagged Slc26a6 from each cotransfection. Western blots depicted in panels A+C were probed with anti-myc antibody R950-25 (1:5000 dilution) and those in panels B+D were probed with anti-HA antibody 71–5500 (1:50 dilution). Panel E: relative expression of primary immunoprecipitated myc-tagged WT Slc26a6 and coimmunoprecipitated HA-tagged WT or mutant Slc26a6. Expression levels were determined by densitometry. To facilitate comparisons between cotransfections (n=3 individual cotransfection events; Tx-1 to Tx-3), protein expression levels determined for the myc-WT/HA-MT cotransfection condition were expressed relative to the densitometry values for the myc-WT/HA-WT cotransfection condition for each experiment, and myc-WT:HA-WT cotransfection expression levels were normalised to 100 (%). *Not significantly different at p=0.3566. tx, transfection; Utx, untransfected.