The Kv7.4-Q580A mutant is less able to bind dynein. (A i) Representative Western blot with HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4-EGFP showing Kv7.4-EGFP bands (∼104 kD). (A ii) Mean data comparing the Kv7.4-EGFP band intensity in the lysates from HEK293B cells transfected with WT-Kv7.4-EGFP or Q580A-Kv7.4–EGFP. (B i) Representative images of PLAs in HEK293B cells transfected with Kv7.4 (left) or Kv7.4-Q580A (right) using Kv7.4 and dynein antibodies. The EGFP is visualized (green) in the insets to show Kv7.4 expression. Each red punctum is representative of the Kv7 channel and dynein protein localizing within 40 nm of one another. (B ii) Quantification of puncta in HEK293B cells (Kv7.4-WT: 15 cells, Kv7.4-Q580A: 20 cells) with Kv7.4 and dynein and the respective antibodies alone as control, according to an unpaired t test. ***, P < 0.0001. Scale bars, 5 µm. Mean values are shown with error bars depicting the SEM.