Figure 5. Western blot analysis of... | Rockefeller University Press

Figure 5.

$Figure 5. Western blot analysis of oocytes expressing WT and S6 mutant Slo2.1 channels. (A) Proteins in the biotinylated membrane fraction were separated by SDS-PAGE electrophoresis, and gels were blotted to detect FLAG-tagged Slo2.1. In the top panel, Slo2.1 monomer is represented by a 120-kD band, which is absent in uninjected oocytes. The 130-kD band in the top panel was detected in all lanes and represents nonspecific labeling by the antibody. For the top panel, lanes 1 and 2 were loaded with samples from oocytes injected with 10 and 1 ng WT FLAG-tagged Slo2.1 cRNA, respectively. For all other panels, lanes 1 and 2 represent samples from oocytes injected with 1 and 10 ng WT FLAG-tagged Slo2.1 cRNA, respectively. Na+/K+ ATPase (plasma membrane marker), but not GAPDH (cytosolic marker), was present in this fraction. (B) Western blots for Na+/K+ ATPase and GAPDH from the cytosolic fraction. (C) Comparison of relative surface protein expression and current magnitude for WT and S6 mutant Slo2.1 channels. Western blots in A were quantified by densitometry using the area under the curve method of ImageJ software to correct for background noise, and the nonspecific 130-kD band observed in all lanes, including the uninjected oocytes lane. The ratio of FLAG-tagged Slo2.1 Na+/K+ ATPase band intensities from the same membrane fraction sample was determined for each lane. The relative band intensity of mutant Slo2.1 proteins (cyan bars) were calculated by normalization to the ratio determined for the sample of oocytes injected with 10 ng WT cRNA. Currents were measured at 0 mV after activation with either 1 mM NFA (ISlo2.1NFA, calculated from data plotted in Fig. 3 B) or by intracellular loading with NaCl (ISlo2.1Na, calculated from data plotted in Fig. 4, C and D).$

Western blot analysis of oocytes expressing WT and S6 mutant Slo2.1 channels. (A) Proteins in the biotinylated membrane fraction were separated by SDS-PAGE electrophoresis, and gels were blotted to detect FLAG-tagged Slo2.1. In the top panel, Slo2.1 monomer is represented by a 120-kD band, which is absent in uninjected oocytes. The 130-kD band in the top panel was detected in all lanes and represents nonspecific labeling by the antibody. For the top panel, lanes 1 and 2 were loaded with samples from oocytes injected with 10 and 1 ng WT FLAG-tagged Slo2.1 cRNA, respectively. For all other panels, lanes 1 and 2 represent samples from oocytes injected with 1 and 10 ng WT FLAG-tagged Slo2.1 cRNA, respectively. Na⁺/K⁺ ATPase (plasma membrane marker), but not GAPDH (cytosolic marker), was present in this fraction. (B) Western blots for Na⁺/K⁺ ATPase and GAPDH from the cytosolic fraction. (C) Comparison of relative surface protein expression and current magnitude for WT and S6 mutant Slo2.1 channels. Western blots in A were quantified by densitometry using the area under the curve method of ImageJ software to correct for background noise, and the nonspecific 130-kD band observed in all lanes, including the uninjected oocytes lane. The ratio of FLAG-tagged Slo2.1 Na⁺/K⁺ ATPase band intensities from the same membrane fraction sample was determined for each lane. The relative band intensity of mutant Slo2.1 proteins (cyan bars) were calculated by normalization to the ratio determined for the sample of oocytes injected with 10 ng WT cRNA. Currents were measured at 0 mV after activation with either 1 mM NFA (I_Slo2.1NFA, calculated from data plotted in Fig. 3 B) or by intracellular loading with NaCl (I_Slo2.1Na, calculated from data plotted in Fig. 4, C and D).