Figure 3. β4 increases surface expression of Slo3.

In A, oocytes from the same batch were injected either with mSlo3::eYFP (eYFP inserted between RCK domains) alone, mSlo3::eYFP+hβ4, or with H₂O. Confocal sections revealed weak fluoresence in oocytes injected only with mSlo3 alone, while hβ4 strongly increased surface fluoresence. For illustrative purposes, an oocyte of each category was positioned together and viewed either with fluorescent (left) or normal illumination (right). In B, oocytes were injected with Slo3-eYFP (eYFP attached to C-terminus) with or without hβ4. As in A, hβ4 substantially increased surface fluorescence. In C and D, Slo3 was FLAG tagged. In C, Slo3 either in samples of total protein or surface protein expressed either alone or together with β4 in Xenopus oocytes was detected with Western blotting. Total Slo3 protein was purified by immunoprecipitation with anti-FLAG M2 agarose. After “blotting” of the immunoprecipitated proteins, 1∶200 anti-Slo3 antibody was used as the primary antibody. Integrated density of the immunoblots was quantified using ImageJ 1.40 g software (http://rsb/info.nih/gov/ij/). For total protein samples, the amount of Slo3 protein in the presence of β4 is 1.2-fold of that in the absence of β4. The corresponding ratio for surface protein is 12. In D, total and surface expression of β4+/−Slo3 are compared. Monoclonal anti-mβ4 antibody (1∶200) served as the primary antibody. The ratio of β4 amount in the presence of Slo3 compared to that without Slo3 is 1 or 11, for total protein or surface protein samples, respectively.