Fig 1: Common features of Kv2.2 and Kv2.1 modulation by Kv6.1.A, current density at +30 mV was measured from indicated subunit combinations (1:1 cDNA ratio of Kv2.2 and Kv6.1 WT or Kv6.1[W416C]) after transient transfection in LM cells. Control groups were cotransfected with soluble GFP to ensure a constant amount of plasmid DNA in the transfection mixture in all experiments. One-way ANOVA followed by Tukey post hoc test was used to compare groups (∗ indicates p < 0.05; ∗∗ indicates p < 0.001), n = 6. B, mOrange-Kv2.2 was coexpressed with Kv6.1, Kv6.1[L284P], and Kv6.1[W416C] in 1:1 ratio for 72 h in HEK293 cells. Whole-cell lysates were separated on SDS-PAGE and probed with anti-Kv2.2 and Na+/K+-ATPase (loading control) antibodies. C, Kv2.2 signal in each group from panel B is plotted as normalized to the Kv2.2 control (channel expressed alone) (n = 3). A repeated measures ANOVA test (on raw Kv2.2 density normalized to β-actin) followed by a post hoc paired t test was used to compare experimental groups (∗ indicates p < 0.05 relative to Kv2.2 alone). D, Kv6.1 or variants were expressed alone or with Kv2.2 (1:1 ratio), for 72 h in HEK293 cells. Whole-cell lysates were separated by phos-tag SDS-PAGE and probed using an anti-GFP (β-actin loading control (n = 3)). Arrows indicate the slower migration of phosphorylated forms of Kv6.1 and Kv6.1[L284P] when coexpressed with Kv2.2 (representative blot from three experiments).