Successively, to investigate the presence of the hypothesized disulfide bridge in the NaCh β1 subunit we applied the diagonal electrophoresis. The results of these bi-dimensional electrophoresis experiments are shown in Fig. 3. The electrophoresis of the samples in the first dimension was done in the absence of any reducing agent in the polyacrylamide gel, while the second-dimension was done in a gel in strong reducing conditions. It is known that
Sunitinib Malate that do not have disulfide bonds run at the same molecular weight under both conditions, placing on a hypothetical diagonal line connecting the opposite corners of the gel. However, those proteins containing intra-molecular disulfide bonds, will run faster on the gel under reducing conditions, as the gyration radius of the polypeptide is strongly modified by their disruption and will be detected as spots outside the diagonal, on the upper-right side of the gel. To confirm the identity of the NaCh β1 subunit, we compared the position on the gel of the spot with the band of 25 kDa of a Western blot of the same sample, revealed with an antibody against the β1 subunit. The comparison of the gels and western blot images containing cell extracts of CHO cell transfected either with α1.4 and WT-β1 or α1.4 and C121W shows that the spot corresponding to WT-β1 subunit lied above the diagonal, whereas when the same experiment was conducted using CHO
cells transfected with α1.4 and C121W β1 subunit, the spot lied out of the diagonal in coincidence with the 25 kDa β1 subunit band on the Western blot (see Fig. 3). Similar experiment done with no transfected CHO cells yielded a clear diagonal without the 25 kDa spot out of the diagonal, as expected (data not shown). We repeated the experiments on three different cell pools for each isoform, with exactly the same results. Therefore, we concluded that the NaCh β1 subunit does present a disulfide bridge, and the pathologic mutation C121W produces a disruption of this covalent interaction.
